Compositions and methods for self-adjuvanting vaccines against microbes and tumors

ABSTRACT

The present invention is drawn to compositions and methods to enhance an immune response in order to prevent or treat infections or hyperproliferative diseases such as cancer. More particularly, the composition is an immunostimulatory intracellular signaling peptide fused directly or indirectly to a peptide that leads to multimerization into complexes of three or more units, where the intracellular signaling peptide must be present in a complex of three or more units in order to stimulate an immune response. Inserting this fusion construct into viruses like HIV-1 or introducing it into dendritic cells or tumor cells is predicted to lead to a positive therapeutic effect in humans, non-human mammals, birds, and fish.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/635,885, filed Sep. 18, 2012, now pending; which is a 35 USC §371National Stage application of International Application No.PCT/US2011/029458 filed Mar. 22, 2011, now expired; which claims thebenefit under 35 USC §119(e) to U.S. Application Ser. No. 61/340,843filed Mar. 23, 2010, now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

GRANT INFORMATION

This invention was made with government support under Grant No. AI63982awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates generally to compositions and methods forimproved immune response to prevent or treat infections orhyperproliferative diseases such as cancer, and more specifically tocompositions and methods for self-adjuvanting vaccines against microbesand tumors.

Background Information

Immune responses are highly desirable when they protect the body frominfection by a microorganism or control the growth of tumors and otherhyperproliferative disorders. To generate an immune response, threecomponents are needed: (1) an antigen; (2) an adjuvant; and (3) adelivery method. The most common means for generating an immune responseis by administering a vaccine. In the case of the tetanus vaccine, theantigen is heat-inactivated tetanus toxoid protein, the adjuvant is alum(i.e., hydrated aluminum potassium sulfate), and the delivery method isthe needle and syringe used for the subcutaneous or intramuscularinjection.

For other kinds of vaccines, notably viral vaccines such as measles,mumps, rubella, polio, and varicella vaccines, a live, replicating virusis used. The live virus is typically attenuated or weakened as part ofits selection and production, but the live virus vaccine contains boththe viral antigens (proteins, carbohydrates, or lipids from the virus)along with the means of delivery. In this case, the delivery componentis intrinsic to the ability of the virus to enter cells, partially orcompletely replicate, and thereby lead to the generation of its antigensin the host. The live virus vaccine can also carry its own adjuvant andthereby elicit a strong immune response. A good example of this is theYellow Fever vaccine 17D in which the live virus vaccine is capable ofinteracting with Toll-Like Receptors (TLRs) on dendritic cells and otherantigen-presenting cells, thereby activating these cells to initiate andamplify an immune response.

On the other hand, some vaccines fail to provide sufficient adjuvantactivity. A good example is the live replicating viral vaccine forRespiratory Syncytial Virus (RSV). About half of all children areinfected by RSV during their first year of life. However, aformalin-fixed RSV virus vaccine failed to protect children. This wassubsequently traced to an inability of the vaccine to provide theappropriate adjuvant activity, specifically the activation of TLRs onantigen-presenting cells. Conversely, if TLR agonists were added to theineffective RSV vaccine, it became sufficient to elicit strongprotective immune responses.

As the example of the RSV vaccine demonstrates, there is a need in thefield to provide an adjuvant when the vaccine alone is insufficient forstimulating antigen-presenting cells. Sometimes this can be provided bymixing an adjuvant with the vaccine, such as mixing the MF59 adjuvantwith the vaccine for influenza. However, this approach is not suitablefor all situations.

In many cases, there is a need for a method to provide the antigen andthe adjuvant co-extensive in space and time. To do this, it ispreferable to incorporate the adjuvant into the vaccine formulation. Forexample, the Yellow Fever 17D vaccine incorporates the viral antigens,the TLR agonist adjuvants, and the delivery method (cell entry mediatedby viral proteins) into a single entity. As such, the antigen andadjuvant are provided co-extensive in space and time.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods to enhance animmune response in order to prevent or treat infections orhyperproliferative diseases such as cancer. More particularly, thecomposition is an immunostimulatory intracellular signaling peptidefused directly or indirectly to a peptide that leads to multimerizationinto complexes of three or more units, where the intracellular signalingpeptide must be present in a complex of three or more units in order tostimulate an immune response.

In one embodiment, the present invention provides an isolated proteinincluding a multimerizing domain operatively joined to a cytoplasmicsignaling domain of a receptor such that the protein assembles into acomplex of three or more protein moieties. In one aspect, the proteinmoieties activate a desired biological effect in cells.

In one aspect, the multimerizing domain includes an N-terminus fragmentor portion of latent membrane protein 1 (LMP1). In another aspect, thecytoplasmic signaling domain includes a cytoplasmic fragment or portionof a member of Tumor necrosis factor receptor superfamily (TNFRSF). Inanother aspect, the cytoplasmic signaling domain includes a cytoplasmicfragment or portion selected from CD40, CD27, Fas, lymphotoxin betareceptor (LTBR), nerve growth factor receptor (NGFR), Tumor necrosisfactor receptor superfamily member 1A (TNFRSF1A), Tumor necrosis factorreceptor superfamily member 1B (TNFRSF1B), Tumor necrosis factorreceptor superfamily member 4 (TNFRSF4), Tumor necrosis factor receptorsuperfamily member 8 (TNFRSF8), Tumor necrosis factor receptorsuperfamily member 9 (TNFRSF9), Tumor necrosis factor receptorsuperfamily member 10A (TNFRSF10A), Tumor necrosis factor receptorsuperfamily member 10B (TNFRSF10B), Tumor necrosis factor receptorsuperfamily member 10D (TNFRSF10D), Tumor necrosis factor receptorsuperfamily member 11A (TNFRSF11A), Tumor necrosis factor receptorsuperfamily member 12A (TNFRSF12A), Tumor necrosis factor receptorsuperfamily member 13B (TNFRSF13B), Tumor necrosis factor receptorsuperfamily member 13C (TNFRSF13C), Tumor necrosis factor receptorsuperfamily member 14 (TNFRSF14), Tumor necrosis factor receptorsuperfamily member 17 (TNFRSF17), Tumor necrosis factor receptorsuperfamily member 18 (TNFRSF18), Tumor necrosis factor receptorsuperfamily member 19 (TNFRSF19), Tumor necrosis factor receptorsuperfamily member 21 (TNFRSF21), or Tumor necrosis factor receptorsuperfamily member 25 (TNFRSF25).

In one aspect, the cell signaling domain includes the cytoplasmic domainof a Latency Membrane Protein 1 (LMP1) of Epstein-Barr virus (EBV) orCD40. In another aspect, the multimerizing domain includes at least fourtransmembrane regions. In another aspect, the multimerizing domainincludes about four to six transmembrane regions. In another aspect, theprotein includes a Latency Membrane Protein 1 (LMP1) or LMP1-CD40 fusionprotein.

In another embodiment, the present invention provides an isolated virus,microbe, or host cell. The isolated virus, microbe, or host cellincludes a first expression cassette for expressing the protein asdescribed herein, and a second expression cassette for expressing anantigen. In one aspect, the isolated virus, microbe, or host cell iswith the proviso that the virus, microbe, or host cell does not includeor contain an Epstein-Barr virus (EBV).

In one aspect, the antigen expressed by the virus, microbe, or host cellgains cell stimulatory activities from the cytoplasmic signaling domain.In another aspect, the protein includes (a) a multimerizing domainincluding an N-terminus fragment or portion of latent membrane protein 1(LMP1); and/or (b) a cytoplasmic signaling domain including acytoplasmic fragment or portion of a member of Tumor necrosis factorreceptor superfamily (TNFRSF).

In one aspect, the protein includes a Latency Membrane Protein 1 (LMP1)or LMP1-CD40 fusion protein. In another aspect, the isolated virus,microbe, or host cell includes a DNA virus or RNA virus. In one aspect,the isolated virus, microbe, or host cell includes a microbe selectedfrom Human immunodeficiency virus-1 (HIV-1), Simian immunodeficiencyvirus (SIV), influenza virus, parainfluenza virus, dengue virus,Hepatitis A virus, Hepatitis B, virus, Hepatitis C virus,Cytomegalovirus (CMV), adenovirus, adeno-associated virus, Simian virus40 (SV40), Modified Vaccinia Ankara (MVA), Vesicular stomatitis virus(VSV), arenaviruses, bunyaviruses, flaviviruses, West Nile virus,Japanese Encephalitis virus, Venezuelan equine encephalitis virus,Eastern equine encephalitis virus, Western equine encephalitis virus,herpesviruses, measles virus, rhabdoviruses, Listeria, Salmonella, orcombinations thereof. In another aspect, the isolated virus, microbe, orhost cell includes a replication-incompetent single-cycle virus. Inanother aspect, the antigen includes a tumor antigen, viral antigen, ormicrobial antigen. In another embodiment, the present invention providesa DNA virus or RNA virus that expresses the isolated protein of claim 1in cells transduced by that virus.

In another embodiment, the present invention provides a vaccinecomposition including (a) an antigen including a tumor antigen, viralantigen, or microbial antigen; (b) an adjuvant including the protein asdescribed herein; and (c) a delivery system including a microorganism.

In one aspect, the protein includes (a) a multimerizing domain includingan N-terminus fragment or portion of latent membrane protein 1 (LMP1);and/or (b) a cytoplasmic signaling domain including a cytoplasmicfragment or portion of a member of Tumor necrosis factor receptorsuperfamily (TNFRSF). In another aspect, the protein includes a LatencyMembrane Protein 1 (LMP1) or LMP1-CD40 fusion protein.

In one aspect, the delivery system includes a DNA virus, RNA virus, orprokaryotic organism. In another aspect, the delivery system includes atumor-associated virus. In another aspect, the delivery system includesa virus selected Human immunodeficiency virus-1 (HIV-1), Simianimmunodeficiency virus (SIV), influenza virus, parainfluenza virus,dengue virus, Hepatitis A virus, Hepatitis B, virus, Hepatitis C virus,Cytomegalovirus (CMV), adenovirus, adeno-associated virus, Simian virus40 (SV40), Modified Vaccinia Ankara (MVA), Vesicular stomatitis virus(VSV), arenaviruses, bunyaviruses, flaviviruses, West Nile virus,Japanese Encephalitis virus, Venezuelan equine encephalitis virus,Eastern equine encephalitis virus, Western equine encephalitis virus,herpesviruses, measles virus, rhabdoviruses, Listeria, Salmonella, orcombinations thereof.

In another embodiment, the present invention provides a method forstimulating an immune response or preventing/treating cancer or aninfectious disease in a subject. The method includes administering to acell an effective amount of a polynucleotide including a firstexpression cassette for expressing the protein as described herein,and/or a second expression cassette for expressing an antigen. If themalignant or infected cell already expresses the targeted antigen(s),then the immune response against that antigen(s) may be stimulated byadministering only the protein as described herein.

In one aspect, at least two nucleic acid sequences are administered,where a first nucleic acid sequence includes the first expressioncassette for expressing the protein as described herein, and a secondnucleic acid sequence includes the second expression cassette forexpressing an antigen. These nucleic acid sequences may be deliveredseparately (e.g., in separate polynucleotide molecules or plasmids) oroperatively linked (e.g., in a single polynucleotide molecule orplasmid). In another aspect, the nucleic acid includes a DNA vaccine. Inanother aspect, the nucleic acid includes an in vitro synthesized andoptionally modified RNA molecule. In one aspect, the nucleic acidincludes (a) a multimerizing domain including an N-terminus fragment orportion of latent membrane protein 1 (LMP1); and/or (b) a cytoplasmicsignaling domain including a cytoplasmic fragment or portion of a memberof Tumor necrosis factor receptor superfamily (TNFRSF). In anotheraspect, the protein includes a Latency Membrane Protein 1 (LMP1) orLMP1-CD40 fusion protein. In one aspect, the antigen includes a tumorantigen, viral antigen, or microbial antigen. In another aspect, thesubject is mammalian. In an additional aspect, the subject is human.

In another embodiment, the present invention provides an avirulent,oncolytic herpes simplex virus having an intact U_(S)12 gene and anendogenous U_(S)11 gene expressed as a late gene, wherein the virus ismodified from the wild-type herpes simplex virus with both γ₁34.5 genesof the virus being deleted and U_(S)11 genes that are expressed asimmediate-early (IE) genes being inserted into the γ₁34.5 gene locus inplace of both γ₁34.5 genes; wherein the virus includes an expressioncassette for expressing the protein as described herein.

In another embodiment, the present invention provides a herpes simplexvirus 1 (HSV1) strain, which is modified such that it lacks one or moreof a functional ICP34.5-encoding gene, a functional ICP6-encoding gene,a functional glycoprotein H-encoding gene and a functional thymidinekinase-encoding gene, and which is derived from HSV1 strain JS1 asdeposited at the European Collection of Cell Cultures (ECACC) underaccession number 01010209; wherein the virus includes an expressioncassette for expressing the protein as described herein.

In another embodiment, the present invention provides a herpes simplexvirus which: (i) includes a gene encoding an immunostimulatory protein;(ii) lacks a functional ICP34.5 encoding gene and a functional ICP47encoding gene; (iii) is replication competent in tumor cells; and (iv)is derived from HSV1 JS1 as deposited at the European collection of cellcultures (ECAAC) under accession number 01010209; wherein the virusincludes an expression cassette for expressing the protein as describedherein.

In another embodiment, the present invention provides a modified,oncolytic herpes simplex virus (HSV) strain including: a modified,oncolytic herpes simplex virus (HSV) strain wherein the HSV strain is aclinical isolate from a recurrent cold sore and has a greater abilitythan a reference laboratory HSV strain modified in the same manner asthe clinical isolate to replicate in or kill tumor cells, and whereinthe reference laboratory HSV strain is selected from the groupconsisting of HSV1 strain 17+, HSV1 strain F and HSV1 strain KOS;wherein the virus includes an expression cassette for expressing theprotein as described herein.

In another embodiment, the present invention provides a method oftreating cancer in subject. The method includes administering to thesubject an effective amount of an oncolytic virus wherein the virusincludes an expression cassette for expressing the protein as describedherein. In one aspect, the oncolytic virus is selected from a NewcastleDisease Virus, a Mumps Virus, a Measles Virus, a Vesicular StomatitisVirus, a Para-influenza Virus, an Influenza Virus, an Adenovirus, aHerpes I Virus, a Vaccinia Virus, a Reovirus, a Seneca Valley virus, anAlphavirus, Sindbis virus, or a combination thereof. In another aspect,the virus is selected from any virus described herein.

In another embodiment, the present invention provides a modified,oncolytic herpes simplex virus 1 strain (HSV1), wherein the oncolytic(HSV1) strain is a clinical isolate from a recurrent cold sore modifiedsuch that it lacks a function ICP34.5-encoding gene, wherein themodified clinical isolate has a greater ability than a referencelaboratory HSV strain modified in the same manner as the clinicalisolate to replicate in or kill tumor cells, and wherein the referencelaboratory strain is selected from the group consisting of HSV1 strain17+, HSV1 strain F and HSV1 strain KOS; wherein the virus includes anexpression cassette for expressing the protein as described herein.

In another embodiment, the present invention provides a herpes viruswhich lacks a functional ICP34.5 encoding gene and which includes two ormore of: (i) a heterologous gene encoding a prodrug converting enzyme;(ii) a heterologous gene encoding a protein capable of causing cell tocell fusion; and (iii) a heterologous gene encoding an immunomodulatoryprotein; wherein the virus includes an expression cassette forexpressing the protein as described herein.

In another embodiment, the present invention provides a method oftreating cancer in a subject in need thereof by administering to a tumorin the subject a therapeutically effective amount of a herpes simplexvirus which: (i) includes an immunostimulatory protein; (ii) lacks afunctional ICP34.5 encoding gene and a functional ICP47 encoding gene;and (iii) is replication competent in infected tumor cells; wherein thevirus includes an expression cassette for expressing the protein asdescribed herein.

In another embodiment, the present invention provides a method ofstimulating an immune response in a human or animal subject, whichmethod includes administering to a subject in need thereof an effectiveamount of an attenuated herpes virus which: (i) lacks a functional vhsgene, or a functional equivalent thereof; (ii) lacks a functional geneencoding ICP47, or a functional equivalent thereof; and (iii) includes afunctional UL43 gene, or a functional equivalent thereof such thatdendritic cells are infected with the virus; wherein the virus includesan expression cassette for expressing the protein as described herein.

In another embodiment, the present invention provides a method ofstimulating an immune response in a human or animal subject, whichmethod includes administering to a subject in need thereof an effectiveamount of an attenuated herpes virus capable of efficiently infecting adendritic cell without preventing antigen processing occurring withinthe infected cell, wherein the virus contains mutations which prevent orminimize the expression of viral immediate early genes in the infectedcell; wherein the virus includes an expression cassette for expressingthe protein as described herein.

In another embodiment, the present invention provides a method ofstimulating an immune response in a human or animal subject, whichmethod includes administering to a subject in need thereof an effectiveamount of an attenuated herpes virus which: (i) lacks a functional vhsgene, or a functional equivalent thereof; (ii) lacks a functional geneencoding ICP47, or a functional equivalent thereof; and (iii) includes afunctional UL43 gene, or a functional equivalent thereof such thatdendritic cells are infected with the virus; wherein the virus includesan expression cassette for expressing the protein as described herein.

In another embodiment, the present invention provides a method ofstimulating an immune response in a human or animal subject, whichmethod includes administering to a subject in need thereof an effectiveamount of an attenuated herpes virus which: (i) lacks a functional vhsgene, or a functional equivalent thereof; (ii) lacks a functional ICP47gene, or a functional equivalent thereof; and (iii) is incapable ofexpressing a substantial amount of functional ICP22, or a functionalequivalent thereof, in mammalian dendritic cells; wherein the virusincludes an expression cassette for expressing the protein as describedherein.

In another embodiment, the invention provides a method of preparing aself-adjuvanting vaccine using a microorganism. In one aspect, themethod uses a microorganism into whose genome has been modified by theintroduction of a multimerizing-intracellular signaling gene cassetteinto the genome of the microorganism. The method includes introducing amultimerizing-intracellular signaling gene cassette into the genome ofthe microorganism. In one aspect, the method further includes deliveringthe genome of the microorganism into a subject. In another aspect, themethod further includes delivering the genetically modifiedmicroorganism into a subject. In an additional aspect, the geneticmodification comprises a multimerizing-intracellular signaling genecassette expresses LMP1 or LMP1-CD40. In another aspect, themultimerizing-intracellular signaling gene cassette expresses LMP1 orLMP1-CD40. In another aspect, the microorganism includes a virus orbacterium. In an additional aspect, the microorganism is Listeria orSalmonella. In another aspect, the microorganism includes an oncolyticvirus. In another aspect, the multimerizing-intracellular signaling genecassette includes a cytoplasmic domain of a death receptor like Fas orTrail receptor. In another aspect, the genome of the microorganism isdelivered into a cell of the subject ex vivo or in vitro. In anotheraspect, the microorganism is a nucleic acid traneferring bacterium. Inanother aspect, the genome of the microorganism is delivered into an armof a human. In another aspect, the microorganism is a pathogen.

In another embodiment, the invention provides the use of the proteindescribed above in the manufacture of a medicament for treatment orprevention of cancer or an infectious disease in a subject. In oneaspect, the protein includes an N-terminus fragment or portion of latentmembrane protein 1 (LMP1) fused with a cytoplasmic signaling domain. Inanother embodiment, the invention provides a protein as described hereinfor use in a method for treatment or prevention of cancer or aninfectious disease in a subject.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter. It should be appreciated that the conception and specificembodiment disclosed may be readily utilized as a basis for modifying ordesigning other structures for carrying out the same purposes of thepresent invention. It should also be realized that such equivalentconstructions do not depart from the invention as set forth in theappended claims. The novel features which are believed to becharacteristic of the invention, both as to its organization and methodof operation, together with further objects and advantages will bebetter understood from the following description when considered inconnection with the accompanying figures. It is to be expresslyunderstood, however, that each of the figures is provided for thepurpose of illustration and description only and is not intended as adefinition of the limits of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawing.

FIGS. 1A-B show the latent membrane protein 1 (LMP1) and LMP1-CD40 asprototypes for a multimerizing-intracellular signaling cassette. FIG. 1Ashows that LMP1 from the Epstein-Barr Virus (EBV) is amembrane-associated protein with an N-terminus that contains sixtransmembrane domains and a C-terminus that includes an intracellularsignaling domain. The LMP1 N-terminus is known to self-associate,leading to the formation of clustered, multimeric patches in the planeof the membrane. FIG. 1B shows that LMP1-CD40 is a chimeric fusionprotein which employs the N-terminus of LMP1 for multimerization andmembrane association and the intracellular signaling domain of CD40. Inone aspect, no spacer or linker is present between the LMP1 and CD40portions, but they can be easily included into the sequence. Both LMP1and LMP1-CD40 are constitutively active, meaning that no ligand or otherstimulus is needed for them to initiate signaling through the CD40pathway.

In LMP1, the 6 transmembrane N-terminal regions enable the formation ofLMP1 clusters in the plasma membrane. This clustering is essential forLMP1 activity. In the LMP1-CD40 fusion protein, the cell signalingC-terminal region of LMP1 has been replaced with the signaling domain ofthe CD40 receptor.

FIG. 2 shows exemplary construction of live, replicating LMP1- andLMP1-CD40-expressing HIV-1. In the proviral plasmid clone, pNL4-3BaL, acoding sequence for enhanced green fluorescent protein (EGFP) can beinserted into the HIV-1 sequence just after the env reading frame andbefore the nef start codon. This is followed by an internal ribosomalentry sequence (IRES) placed just before the nef sequence, such that Nefprotein is also produced. As a consequence of this design, a live,replicating virus is encoded by this nucleic acid sequence (Levy et al.(2004) Proc Natl Acad Sci USA 101: 4204-9). In the present invention,the EGFP coding sequence is replaced with either LMP1 or LMP1-CD40. Ascontrols, secreted multimeric soluble forms of CD40L formed by fusionwith surfactant protein D (SP-D-CD40L), a variant termed SP-D-CD40L-NST,and a secreted multimeric soluble form of Glucocorticoid-induced tumornecrosis factor receptor ligand (GITRL) are also introduced into theEGFP site.

FIG. 3 shows replication in and stimulation of human monocyte-derivedmacrophages by LMP1- and LMP1-CD40-expressing HIV-1. Referring to FIG.2, three plasmids are used: one for the unmodified proviral plasmidclone, pNL4-3BaL, containing the EGFP sequence just 5′ to the IRES-nefportion of the sequence; the same plasmid except replacing the EGFPsequence with LMP1 just 5′ to the IRES-nef portion of the sequence; andthe same plasmid except replacing the EGFP sequence with LMP1-CD40 just5′ to the IRES-nef portion of the sequence. To produce live virus fromthese plasmids, the plasmids are transfected into 293T cells using thecalcium phosphate method. Forty-eight hours later, virus containingsupernatants are harvested and used to infect human monocyte-derivedmacrophages (MDM) in culture. By flow cytometry 48 hours post infection,about 10% of the MDM became EGFP positive, an indicator of cellularinfection. At the same time, supernatants are harvested and assayed byELISA for p24 Gag (as a measure of new progeny virus production), thechemokine macrophage inflammatory protein 1-beta (MIP-1β also designatedCCL4), and interleukin-8 (IL-8) which is a cytokine. As shown in thefigure, progeny virus (p24) is best produced by macrophages infected bythe original pNL4-3/BaL clone (labeled “HIV only” in the figure) and toa lesser extent by macrophages infected with the HIV-1 viruses carryingLMP1 (labeled “HIV-LMP1”) or LMP1-CD40 (labeled “HIV-LMP1-CD40”).However, the original pNL4-3/BaL clone (labeled “HIV only”) does nottrigger the production of MIP-1β and the infected macrophages producedonly small amounts of IL-8. In contrast, macrophages infected by theLMP1 containing virus (labeled “HIV-LMP1”) produces large amounts ofMIP-1β and IL-8 (outside the range of the ELISA) whereas macrophagesinfected by the LMP-CD40 containing virus (labeled “HIV-LMP1”) producedno detectable MIP-1β and above background levels of IL-8. Thisexperiment demonstrates the surprising result that a virus like HIV-1that normally does not stimulate target cells like macrophages can beengineered to stimulate these cells if amultimerization-intracytoplasmic signaling cassette (e.g., LMP1 orLMP1-CD40) is inserted into and expressed by the virus. In this example,the resulting engineered virus can still replicate in its target cells.

FIG. 4 shows further studies on the stimulation of humanmonocyte-derived macrophages by LMP1- and LMP1-CD40-expressing HIV-1. Ina subsequent study similar to FIG. 3 above, monocyte-derived macrophagesare infected with virus and cultured for 9 days until cellular infectionis well-established. Supernatants are collected and cytokines aremeasured. As shown, NL4-3/BaL virus engineered to express LMP1(“NL4-Bal-LMP1”) stimulates macrophages to produce IL-8, IL-1β, IL-6,IL-12p70, and TNFα, but does not increase the production of IL-10, whichis an immunosuppressive cytokine. NL4-3/BaL virus engineered to expressLMP1-CD40 (“NL4-Bal-LMP1-CD40”) has a similar effect on macrophages inculture, with some fine differences noted. As controls, NL4-3/BaL virusexpressing EGFP (“NL4-Bal-EGFP”) and wild-type NL4-3/BaL virus(“NL4-Bal”) have similar effects on macrophages as media alone (“Mock”).

FIG. 5 shows stimulation of human monocyte-derived dendritic cells byLMP1- and LMP1-CD40-expressing HIV-1. Dendritic cells (DCs) areconsidered to be the primary antigen-presenting cell for T cells. Toproduce DCs for study, blood monocytes are isolated from human venousblood and cultured in GM-CSF/IL-4 for 6 days by standard methods bywhich time they have differentiated into DCs. Then the cultured DCs areinfected with various forms of the NL4-3/BaL clone of HIV-1 at amultiplicity of infection (MOI) of 0.1 and the culture supernatant iscollected 9 days later and assayed for the presence of cytokines.Bacterial lipopolysaccharide (LPS) from E. coli B011 (100 ng/ml) is usedas a positive control. In human DCs, LMP1 is able to stimulate theproduction of IL-8, IL-1β, TNFα, and IL-6, whereas LMP1-CD40 is onlyable to stimulate the production of IL-8. Although the virus used hereis capable of replicating in other target cells, there is littleproduction of progeny virus as measured by the release of p24 Gag intothe supernatant. The surprising result of these data is that acomposition of the present invention comprised of amultimerization-intracellular signaling cassette can be used tostimulate DCs, a key type of antigen-presenting cell.

FIG. 6 shows further studies on the stimulation of humanmonocyte-derived dendritic cells by LMP1- and LMP1-CD40-expressingHIV-1. As in FIG. 5, DCs are infected with various NL4-3/BaL HIV-1 viralconstructs. Controls include virus engineered to express the viral clonelacking EGFP (“pNL4-Bal”) or a secreted form of multimeric CD40L(SP-D-CD40L) abbreviated “pNL4-BaL-CD40L” or a similar protein(SP-D-CD40L-NST wherein the stalk region of CD40L is absent and a humantPA signal sequence is used) abbreviated “pNL4-BaL-CD40L-NST”. In thisexperiment, the positive control is MIMIC™ (Clontech, Palo Alto,Calif.), a cytokine mix designed to stimulate DCs. As shown, theLMP1-expressing virus stimulated DCs to produce IL-8, IL-1β, TNFα,IL-12p70, and IL-6. In contrast, the LMP1-CD40-expressing virusstimulates DCs to produce IL-6. Neither the LMP1- nor theLMP1-CD40-expressing virus induced DCs to produce IL-10, animmunosuppressive cytokine, at levels above background.

FIGS. 7A-B shows that LMP1-CD40-expressing HIV-1 stimulates DCs topresent antigen. The mixed leukocyte reaction (MLR) is a classic test ofthe presentation and response to foreign antigens. Consequently,dendritic cells are infected with the various HIV-1 constructs for 5days. Then peripheral blood mononuclear cells (PBMCs) from a seconddonor are added along with nevirapine to prevent HIV-1 infection fromspreading to the added CD4+ T cells. After 5 days of MLR culture, the Tcells are stained for either CD4 (FIG. 7A) or CD8 (FIG. 7B) and analyzedfor intracellular ki67 as a marker for entry into the cell cycle andproliferation. As shown, the most prominent effects in theMLR-responding population are in CD4+ and CD8+ T cells co-cultured withDCs exposed to the LMP1-CD40-expressing HIV-1 virus. Controls usingHIV-1 that expresses soluble multimeric forms of CD40L (SP-D-CD40L andSP-D-CD40L-NST) or GITRL (SP-D-GITRL) also have some stimulatoryeffects. * p<0.05, ** p<0.01, *** p<0.001 compared to control virusNL4-3/BaL-EGFP.

FIG. 8 shows that construction of a single-cycle SIV clone (scSIV) thatexpresses LMP1 or LMP1-CD40. The figure shows the design of a proviralclone of Simian Immunodeficiency Virus (SIV) in which inactivatingmutations have been introduced into codons for the Protease (PR) andIntegrase (IN) genes. The diagram shows how other plasmids (encodingVSV-G envelope and a Gag-Pol polyprotein) are used to preparepseudotyped virus that in turn is used to introduce the construct into293T cells. As a result of this process, the 293T release recombinantvirus that can infect SIV-susceptible cells yet not replicate in themdue to a lack of PR and IN. On the right are shown the cassettes thatare introduced into the nef-spliced mRNA in place of Nef: EGFP; LMP1;LMP1-CD40; SP-D-GITRL; SP-D-CD40L; and SP-D-CD40L-NST.

FIG. 9 shows stimulation of human dendritic cells by scSIV expressingLMP1 or LMP1-CD40. Human monocyte-derived dendritic cells (DCs) areinfected with scSIV virus expressing LMP1, LMP1-CD40, or controls at 50ng p27 Gag protein per million cells. As above, MIMIC™ is a standardcytokine cocktail used as a positive control. Supernatants are collectedafter 5 days and cytokines measured. As shown, the LMP1-expressing scSIVstimulates DCs to produce IL-8, IL-1β, TNFα, IL-12p70, and IL-6, butdoes not stimulate the production of IL-10, an immunosuppressivecytokine. LMP1-CD40-expressing scSIV stimulates DCs to produce IL-8,TNFα, and IL-6, but not TNFα, IL-12p70, or IL-10.

FIG. 10 shows self-adjuvanticity of a single-cycle SIV expressing theLMP1 multimerizing-intracellular signaling cassette. The presentinvention provides that single-cycle SIV (scSIV) can carry its ownantigens into immune responses and a multimerizing-intracellularsignaling cassette can act as an adjuvant for this. Humanmonocyte-derived dendritic cells (DCs) are studied in vitro. The DCs areexposed to the scSIV viruses for 4 days, and then co-cultured with humanT cells for another 12 days. After the co-culture period, the cells aretransferred to the wells of an Enzyme Linked Immuno-Spot (ELISPOT) plateand stimulated with a pool of SIV Gag 15-mer peptides. This assayprovides a rigorous evaluation of antigen-presenting function and T cellresponses, given that the T cells are naïve for Gag antigen on day 0. Asshown in the figure, scSIV-LMP1 induces a significant increase inGag-specific interferon gamma responsive T cells (p<0.01 comparingscSIV-LMP1 to scSIV-EGFP). These data and similar results with HIV-LMP1demonstrate that scSIV-LMP1 is immunostimulatory and effectivelyself-adjuvanting by the inclusion of the LMP1multimerizing-intracytoplasmic signaling cassette. This surprisingresult is the prototype for a range of viruses, vectors, and tumor cellswhere the inclusion of a multimerizing-intracytoplasmic signalingcassette is predicted to induce a strong immune response to the antigensthat are co-extensive in space and time.

FIG. 11 shows introducing LMP1 into Modified Vaccinia Ankara byhomologous recombination. The schematic shows a transfer vector modifiedfrom pLW-44 in which the Vaccinia mH5 promoter drives the expression ofLMP1.

FIGS. 12A-B shows that LMP1 or LMP1-CD40 mRNAs alone are sufficient toactivate DCs. LMP1, LMP1-CD40, or control GFP mRNAs are introduced intoDCs by electroporation. Other controls are DC cultures electroporatedwithout adding mRNA (mock) and non-transfected, untreated DCs. Following48 hours of culture, cell surface markers are analyzed by flow cytometryand cytokine production is analyzed by cytometric bead analysis (CBA).(*, P 0.05; **, P 0.01; ***, P 0.001 using an unpaired t-test comparedto the GFP mRNA control). FIG. 12A shows that LMP1 or LMP1-CD40 mRNAtransduction upregulates CD40 and CD83 maturation markers and CD80 andCD86 co-stimulatory molecules as measured by mean fluorescence intensity(MFI). Bars show the means and SEM of triplicate wells. FIG. 12B showsthat LMP1 or LMP1-CD40 mRNA transduction upregulates the secretion ofcytokines IL-6, IL-8, and TNFα, but is unable to upregulate secretion ofIL-1β, IL-10, or IL-12p70. Bars show the means and SEM of triplicatewells.

FIGS. 13A-B shos that HIV-LMP1 or HIV-LMP1-CD40 viral transductionactivates DCs and macrophages to express cell maturation- andactivation-associated surface molecules. FIG. 13A shows the expressionof surface markers on DCs 4 days after viral transduction. Flowcytometry events are first gated for DCs using forward scatter and sidescatter. Isotype antibody control staining (grey filled histogram),HIV-GFP (thin line), or HIV-LMP1 and HIV-LMP1-CD40 (thick lines) areshown. Viral transduction with HIV-LMP1 results in dendritic cellactivation and maturation as measured by increased levels of CD40, CD80,CD86, CD83, and CCR7 expression. By comparison, viral transduction withHIV-LMP1-CD40 produces a modest increase in CD40 and CD83 expression anda minimal increase in CD80 and CCR7 expression when compared to HIV-GFPtransduced cells. FIG. 13B shows the expression of surface markers onmacrophages 4 days after viral transduction. Flow cytometry events arefirst gated for DCs using forward scatter and side scatter. Isotypeantibody control staining (grey filled histogram), HIV-GFP (thin line),or HIV-LMP1 and HIV-LMP1-CD40 (thick lines) are shown. HIV-LMP1transduction activated macrophages to express higher levels of CD40 andCD83 maturation markers and CD80 and CD86 co-stimulatory molecules. Bycomparison, viral transduction by HIV-LMP1-CD40 is less active andinduced increased levels of CD83 and CD86, but not CD40 or CD80. Theseresults are representative of three experiments on three differentdonors.

FIGS. 14A-B shows that HIV-LMP1 or HIV-LMP1-CD40 viral transductionactivates DCs and macrophages to secrete immunostimulatory cytokines.FIG. 14A shows the effects of viruses on cytokine secretion by DCs. DCsare exposed to viruses at an MOI of 0.1, cultured for 7 d, andsupernatants are collected for analysis by cytokine bead array (CBA)assay. As shown, HIV-LMP1 induces significant increases in IL-6 and IL-8secretion, modest increases in IL-1β, IL-12p70 and TNFα secretion, andno induction of IL-10 secretion. By comparison, HIV-LMP1-CD40 inducessignificant increase in IL-6 secretion, reflecting fine differences inthe cell signaling induced by the LMP1 vs. LMP-CD40 adjuvant cassettesin DCs. The bars show the means from three independent experiments withDCs from three different donors (*, P 0.05; **, P 0.01; ***, P 0.001using an unpaired t-test compare to the HIV-GFP control virus). FIG. 14Bshows the effects of viruses on cytokine secretion by macrophages. Thesecells are exposed to viruses at an MOI of 0.1 and supernatants arecollected at various time points for CBA cytokine analysis. Formacrophages, HIV-LMP1 induces a significant increase in secretion ofIL-1β, IL-6, IL-8, IL-12p70 and TNFα, but little or no increase in IL-10production. By comparison, HIV-LMP1-CD40 induces a significant increasein IL-1β, IL-6, IL-8 and TNFα secretion. The bars show the means fromthree independent experiments with DCs from three different donors.

FIGS. 15A-B shows that HIV-LMP1 enhances the ability of DCs to presentHIV Gag antigen to T cells in an in vitro immunization assay. FIG. 15Ashows a schematic of the experimental protocol. DCs from an HIVseronegative donor are exposed to different HIV viruses for 6 days,washed, and then incubated with autologous T cells for 12-days in thepresence of nevirapine and IL-2 (5 U/ml) starting on day 3. Cultures arethen restimulated with a consensus clade B 15-mer Gag peptide pool andIFN-γ ELISPOT analysis is performed 24 hours later. FIG. 15B shows thatDCs exposed to native HIV infection do not stimulate anti-HIV T cellresponses. This is consistent with the known inability of HIV tostimulate strong T cell responses when compared to more effective viralvaccines for other viruses. However, HIV-LMP1 strongly enhances anti-GagT cell responses (***, P<0.001 by unpaired t-test comparing HIV-LMP1with the HIV-GFP control virus). This shows the ability of the LMP1adjuvant gene cassette to convert a poorly immunogenic virus into astrongly immunogenic one. By comparison, HIV-LMP1-CD40 is much lessactive in this assay, consistent with the overall weaker effect ofHIV-LMP1-CD40 in DCs compared to HIV-LMP1 virus. These results arerepresentative of three experiments on three different donors.

FIG. 16 shows that LMP1 and LMP1-CD40 proteins are expressed by HIV-LMP1and HIV-LMP1-CD40 constructs. To show that the insertion of LMP1 andLMP1-CD40 coding sequences into HIV leads to translated proteins,proviral DNA plasmids for HIV-LMP1 or HIV-LMP1-CD40 are transfected into293 cells along with the HIV-GFP control construct. Cells are lysed 24hours later, processed by SDS PAGE followed by transfer ontonitrocellulose membranes and staining with antibodies for Gag (upperpanels), LMP1 N-terminus (middle panels), or the C-terminalintracytoplasmic domain of CD40 (lower panels). As shown, cellstransfected with all three viral constructs express p24 Gag. The LMP1protein is detected in cells transfected with the LMP1 constructs. Theintracytoplasmic signaling domain of CD40 can be observed inLMP1-CD40-transfected cells.

FIG. 17 shows that HIV-LMP1 or HIV-LMP1-CD40 induces chemokineproduction in macrophages. Macrophages are infected for 7 days and thenanalyzed by RT-PCR to measure steady-state levels of CCL3 (MIP-1α), CCL4(MIP-1β), and CCL5 (RANTES). HIV infection is known to induce β (CC)chemokines under certain conditions, but HIV-LMP1-CD40 infection inducedincreased levels of these chemokines. By comparison, HIV-LMP1 infectionis less immunostimulatory for macrophages chemokine production.

HIV-LMP1 and HIV-LMP1-CD40 infection induces activation-associatedmorphological changes in DCs and macrophages. DCs and macrophages areinfected with HIV wild-type, HIV-EGFP, HIV-LMP1 or HIV-LMP1-CD40 at anMOI of 0.1. Four days later, the cells are photographed using aninverted phase contrast microscope. DCs infected by HIV-LMP1 orHIV-LMP1-CD40 develop elongated dendrite-like processes. Macrophagesinfected by these viruses, but not the control HIV or HIV-GFP viruses,undergo extensive clumping. The degree of clumping is less withHIV-LMP1-CD40 than with HIV-LMP1, suggesting that HIV-LMP1-CD40 is lessactive than HIV-LMP1 in macrophages.

FIG. 18 shows that plasmids encoding LMP1 (pLMP1) or LMP1-CD40(pLMP1-CD40) can be used as DNA vaccine adjuvants. As DNA vaccineadjuvants, pLMP1 and pLAMP1-CD40 can help to protect mice from melanomametastases. As shown in FIG. 18, pSP-D-CD40L is also very effective.These data show that stimulating cells from within using a clusteredCD40 receptor (LMP1 or LMP1-CD40) is nearly as effective as stimulatingcells from without using a multimeric soluble CD40L construct.

In another aspect, C57BL/6 mice are either untreated (naïve) or DNAvaccinated intramuscularly with 50 μg plasmid DNA encoding melanomaantigen pgp100 combined with 50 μg either control empty vector plasmid(pcDNA3.1) or pLMP1, pLMP1-CD40, or pSP-D-CD40L dissolved in a totalinjected volume of 100 μL of phosphate-buffered saline. Vaccinations aredelivered every two weeks for three doses. One week after the lastvaccination, the mice are injected intravenously with 1×10⁵ B16F10melanoma cells. Twenty-one (21) days later, the mice are euthanized; thelungs were removed, and examined for lung metastases. The resultsindicate that the pgp100+pcDNA3.1 control has no protection, wherepgp100+pLMP1 and pgp100+ pLMP1-CD40 completely protected 2 of 5 and 3 of5 mice, respectively. This demonstrates that plasmids encoding LMP1(pLMP1) or LMP1-CD40 (pLMP1-CD40) can serve as immune adjuvants for DNAvaccines, in this case a tumor DNA vaccine.

FIG. 19 shows an exemplary single cycle SIV (scSIV) viral genome of theinvention. The parent vector, expressing GFP from the Nef promoter, iscloned by overlap PCR and inserted into the SIVmac239 FS-ΔPR-ΔINEGFPvector using unique XbaI and SacII sites. To create immunostimulatoryforms of scSIV, LMP1 or LMP1-CD40 is inserted in place of the GFP geneas shown.

FIG. 20 shows an exemplary Western blot of 293T cell lysates transfectedwith SIV expressing LMP1 or LMP1-CD40, where SIV virus expressing LMP1or LMP1-CD40 induces morphological and other changes in DCs andmacrophages. Virus expressing GFP serves as a negative control. Blotsare stained with anti-Gag (upper panels), anti-LMP1 intracellular domain(middle panels), or anti-CD40 intracellular domain (lower panels). Gagp27 is present in all lysates. LMP1 and CD40 intracellular domains arepresent only in cells transfected with LMP1 or LMP1-CD40 viralconstructs respectively. Only LMP1 or LMP1-CD40 expressing virusesinduce elongation of human DCs or macrophages, suggesting the activationand maturation of cells in the culture.

FIGS. 21A-B shows that viral transduction of human DCs or macrophageswith scSIV expressing LMP1 or LMP1-CD40 results in increased levels ofmaturation and activation markers. The expression levels of surfacemarkers from three independent experiments are presented as meanfluorescence intensity (MFI). FIG. 21A shows that the expression ofsurface markers on SIV infected DCs is examined by flow cytometry 4 daysafter transduction. Transduction with scSIV-LMP1 results in dendriticcell activation and maturation as measured by significantly increasedlevels of CD40, CD80 and CD83 expression, while scSIV-LMP1-CD40 resultsin significant increased levels of CD40, CD80 and HLA-DR expression whencompared to scSIV-GFP-transduced cells. FIG. 21B shows that theexpression level of surface markers on scSIV virus-transducedmacrophages is examined 4 days after transduction by flow cytometry froma representative donor. Transduction with scSIV-LMP1 results inincreased levels of CD40 and CD80 expression, while scSIV-LMP1-CD40results in increased levels of CD40, CD80 and CD83 expression comparedto scSIV-GFP-transduced macrophages. Data are analyzed with the unpairedt test: *, p<0.05; **, p<0.01; ***, p<0.001 compared with the scSIV-GFPinfected group.

FIGS. 22A-B shows that scSIV expressing LMP1 or LMP1-CD40 inducesincreased secretion of inflammatory cytokines and β-chemokines, andcorrespondingly increases the level of their steady-state mRNAs. Humaninflammatory cytokine quantitation is performed by cytometric bead array(CBA). Cytokine concentrations from three independent experiments arepresented. Data are analyzed with the unpaired t test: *, p<0.05; **,p<0.01; ***, p<0.001 compared with the scSIV-GFP infected group. MIP-1α,MIP-1β, and RANTES mRNA expressions are analyzed by real-time RT-PCRassay. FIG. 22A shows that DCs are infected with the various SIV virusesat MOI of 0.05 and supernatants are collected at various time intervals.Virus expressing LMP1 results in a significant increase in IL-1β, IL-6,IL-8, IL-10, IL-12p70 and TNFα, while LMP1-CD40 results in an increasein IL-1, IL-6, IL-8, IL-10 and TNFα at various time points postinfection. No measurable amounts of IL-12p70 are detected in GFP andLMP1-CD40 groups. FIG. 22B shows that macrophages are infected withdifferent scSIV viruses for 4 days. Total cellular RNA is isolated,reverse transcribed to cDNA and MIP-1α, MIP-1β, and RANTES mRNAexpressions are analyzed by real-time PCR assay. Virus expressing LMP1results in significant increase in MIP-1β and RANTES mRNA expression,whereas LMP1-CD40 results in significant increase in MIP-1α, MIP-1β andRANTES mRNA expression. Expression of GAPDH is used for normalization ofsamples.

FIGS. 23A-B shows that LMP1 induces enhanced T cell responses in a Gagpeptide-specific IFN-γ ELISPOT assay. FIG. 23A shows a schematicillustration of an experimental protocol of the invention. DCs from anHIV seronegative donor are transduced with scSIV viruses for 4 days,washed, and then incubated with autologous T cells for 12-days in thepresence of nevirapine and IL-2 (5 U/ml) starting on day 3 of thecoculture. After 12 days, cultures are restimulated with a consensusSIVmac239 15-mer Gag peptide pool and IFN-γ ELISPOT analysis isperformed 24 hours later. FIG. 23B shows that DCs infected with parentscSIV are unable to stimulate anti-SIV T cell responses, while thenef-deleted virus scSIV-GFP induced a modest T cell response. DCinfected with scSIV-LMP1 and LMP1-CD40 can significantly enhanceanti-Gag T cell responses (p<0.001). Results are representative of threeindependent experiments using three different donor blood samples.

FIG. 24 shows that HIV-1 lentiviruses carrying LMP1 or LMP-1CD40 canactivate human DCs in vitro. Cytokines profiles including IL-12p70,TNFα, IL-6, IL-1β, and IL-8 are illustrated.

FIGS. 25A-B shows that HIV-LMP1 and HIV-LMP1-CD40 can activate theantigen-presenting function of human DCs. FIG. 25A shows APC functionmeasured as allopresentation in MLR. DCs are transduced with HIV aloneor HIV expressing EGFP, LMP1, LMP1-CD40, or SP-D-CD40L and cultured for6 days. In the presence of neviapine, allogeneic T cells are added(1:10) and cultured for another 5 days. APC function is measured by theproliferation of these added T cells as judged by Ki67 staining.HIV-LMP1 augmented CD4+ T cell responses and HIV-LMP1-CD40 augmentedboth CD4+ and CD8+ T cell responses. FIG. 25B shows APC functionmeasured in an in vitro immunization experiment. Using blood from anHIV-negative donor, DCs are transduced with HIV viruses for 6 days,washed, and then incubated with autologous T cells for 12-days in thepresence of 5 U/ml of IL-2 from day 3. The cells are then transferred toELISPOT plates and restimulated with a 15-mer Gag peptide pool andassayed for IFN-γ spots. Compared to HIV-EGFP, HIV-LMP1 induces astatistically significant increase in de novo T cell responses(p<0.001).

FIG. 26 shows calibrating infectivity and optimization of multiplicityof infection (MOI) of scSIV. To calculate the optimal infection dose,CEM cells are infected with a range of ng/million cells of VSV-Gpseudotyped scSIV for 4 days and then stained with FITC anti-p27antibody and analyzed by flowcytometry. Optimal infectivity can beobserved at 50 ng scSIV per million cells (MOI of 0.05).

FIG. 27 shows enhanced dendritic cell (DC) maturation by ModifiedVaccinia Ankara (MVA) virus containing LMP1 or LMP1-CD40 adjuvant genecassette. Monocyte-derived dendritic cells (DCs) are prepared byculturing monocytes for 6 days in GM-CSF+IL-4 by standard methods.Recombinant MVA is prepared using the pLW-44 homologous transfer vectorthat expresses Green Fluorescent Protein (GFP) from the Vaccinia P11promoter and a second gene from the Vaccinia mH5 promoter. By insertingLMP1 or LMP1-CD40 into the pLW-44 plasmid and then using homologousrecombination to transfer LMP1 or LMP1-CD40 into MVA three kinds ofrecombinant viruses are made: MVA expresses only GFP (recombined withunmodified pLW-44), GFP+LMP1 (recombined with pL-44 containing LMP1 asthe second gene), or GFP+LMP1-CD40 (recombined with pLW-44 containingLMP1-CD40 as the second gene). DCs are transduced with these viruses ata multiplicity of infection (MOI) of 10, cultured for 48 hours, and thenanalyzed by flow cytometry using phycoerythrin-conjugated anti-CD83antibody to stain for the CD83 maturation marker. Uninfected resting DCsdo not express GFP and express low levels of the CD83 maturation marker.Following infection by MVA expressing only GFP, transduced DCs expressboth GFP and a low percentage are positive for CD83. In contrast,following infection by MVA expressing both GFP and LMP1 or LMP1-CD40,transduced DCs express both GFP and a higher percentage are positive forCD83. This demonstrates the additive effect of LMP1 or LMP1-CD40 on MVAstimulation of DCs to mature.

FIG. 28 shows proposed DC-NILV vaccines using LMP1 or LMP1-CD40 based onpreviously disclosed vaccines. The FOVA construct has been disclosed inYang et al. (2008) Nature Biotechnology 26:326-334, and the FUWGagconstruct has been disclosed in Dai Bingbing et al. (2009) Proc NatlAcad Sci 106:20382-20387.

FIG. 29 shows an illustration of TNF Receptor SuperFamily and commonfeatures among some of the members. Arrows indicate cleavage sites ofTurin or other proteases. TNF homology domains (THD; shaded boxes),collagen domains (clear boxes), cycsteine-rich domains (dark and shadedboxes), and death domains (dark boxes close to the bottom of the figure)are illustrated.

DETAILED DESCRIPTION OF THE INVENTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to particularcompositions, methods, and experimental conditions described, as suchcompositions, methods, and conditions may vary. It is also to beunderstood that the terminology used herein is for purposes ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyin the appended claims.

It is readily apparent to one skilled in the art that variousembodiments and modifications can be made to the invention in thisapplication without departing from the scope and spirit of theinvention.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Still further, the terms“having,” “including,” “containing,” and “comprising” areinterchangeable and one of skill in the art is cognizant that theseterms are open ended terms.

The term “adjuvant” refers to an immunostimulant that triggersactivation of antigen-presenting cells such as dendritic cells,macrophages, and B cells. Adjuvants are also understood to provide a“danger” signal indicating that the immune system should go into a stateof alert. Without an adjuvant, immune responses may either fail toprogress or may be diverted into ineffective immunity or tolerance.Adjuvants are often needed for effective preventative or therapeuticvaccines, or for inducing an anti-tumor immune response.

The term “allogeneic” as used herein, refers to cell types or tissuesthat are antigenically distinct. Thus, cells or tissue transferred fromthe same species can be antigenically distinct.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. An antigen can be derived from organisms, subunits ofproteins/antigens, killed or inactivated whole cells or lysates.Exemplary organisms include but are not limited to, Helicobacters,Campylobacters, Clostridia, Corynebacterium diphtheriae, Bordetellapertussis, influenza virus, parainfluenza viruses, respiratory syncytialvirus, arenaviruses, bunhaviruses, flaviviruses, filoviruses, West Nilevirus, Japanese Encephalitis virus, Venezuelan equine encephalitisvirus, Eastern equine encephalitis virus, Western equine encephalitisvirus, Borrelia burgdorfei, Plasmodium, herpesviruses, humanimmunodeficiency virus, papillomavirus, Vibrio cholera, E. coli, measlesvirus, rotavirus, mycobacteria, staphylococci, streptococci, shigella,Salmonella typhi, and Neisseria gonorrhea. Therefore, a skilled artisanrealizes that any macromolecule, including virtually all proteins orpeptides, can serve as antigens. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan realizes that anyDNA, which contains nucleotide sequences or partial nucleotide sequencesof a pathogenic genome or a gene or a fragment of a gene for a proteinthat elicits an immune response results in synthesis of an antigen.Furthermore, one skilled in the art realizes that the present inventionis not limited to the use of the entire nucleic acid sequence of a geneor genome. It is readily inherent that the present invention includes,but is not limited to, the use of partial nucleic acid sequences of morethan one gene or genome and that these nucleic acid sequences arearranged in various combinations to elicit the desired immune response.

The term “antigen-presenting cell” is any of a variety of cells capableof displaying, acquiring, or presenting at least one antigen orantigenic fragment on (or at) its cell surface. In general, the term“antigen-presenting cell” can be any cell that accomplishes the goal ofthe invention by aiding the enhancement of an immune response (i.e.,from the T-cell or -B-cell arms of the immune system) against an antigenor antigenic composition. Such cells can be defined by those of skill inthe art, using methods disclosed herein and in the art. As is understoodby one of ordinary skill in the art, and used herein certainembodiments, a cell that displays or presents an antigen normally orpreferentially with a class II major histocompatibility molecule orcomplex to an immune cell is an “antigen-presenting cell.” In certainaspects, a cell (e.g., an APC cell) may be fused with another cell, suchas a recombinant cell or a tumor cell that expresses the desiredantigen. Methods for preparing a fusion of two or more cells are wellknown in the art. In some cases, the immune cell to which anantigen-presenting cell displays or presents an antigen is a CD4+ T cellor a CD8+ T cell. Additional molecules expressed on the APC or otherimmune cells may aid or improve the enhancement of an immune response.Secreted or soluble molecules, such as for example, cytokines andadjuvants, may also aid or enhance the immune response against anantigen. Such molecules are well known to one of skill in the art, andvarious examples are described herein.

The term “cancer” as used herein is defined as a hyperproliferation ofcells whose unique trait—loss of normal controls—results in unregulatedgrowth, lack of differentiation, local tissue invasion, and metastasis.Examples include but are not limited to, melanoma, non-small cell lung,small-cell lung, lung, hepatocarcinoma, leukemia, retinoblastoma,astrocytoma, glioblastoma, gum, tongue, neuroblastoma, head, neck,breast, pancreatic, prostate, renal, bone, testicular, ovarian,mesothelioma, cervical, gastrointestinal, lymphoma, brain, colon,sarcoma or bladder.

The terms “cell,” “cell line,” and “cell culture” as used herein may beused interchangeably. All of these terms also include their progeny,which are any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.

As used herein, the term “co-extensive in space and time” refers to theconcordance of two functional domains in the same cellular environmentat the same time. More particularly, it refers to the concordance of anantigen and an adjuvant in the same cellular environment at the sametime. Most especially, it refers to a self-adjuvanting construct thatassociates an antigen or antigens with an adjuvant in the same cellularenvironment at the same time.

As used herein, the term “cDNA” is intended to refer to DNA preparedusing messenger RNA (mRNA) as template. The advantage of using a cDNA,as opposed to genomic DNA or DNA polymerized from a genomic, non- orpartially-processed RNA template, is that the cDNA primarily containscoding sequences of the corresponding protein. There are times when thefull or partial genomic sequence is preferred, such as where thenon-coding regions are required for optimal expression or wherenon-coding regions such as introns are to be targeted in an antisensestrategy.

The term “dendritic cell” (DC) is an antigen-presenting cell existing invivo, in vitro, ex vivo, or in a host or subject, or which can bederived from a hematopoietic stem cell or a monocyte. Dendritic cellsand their precursors can be isolated from a variety of lymphoid organs,e.g., spleen, lymph nodes, as well as from bone marrow and peripheralblood. The DC has a characteristic morphology with thin sheets(lamellipodia) extending in multiple directions away from the dendriticcell body. Typically, dendritic cells express high levels of majorhistocompatibility complex (MHC) and costimulatory (e.g., B7-1 and B7-2)molecules. Dendritic cells can induce antigen specific differentiationof T cells in vitro, and are able to initiate primary T cell responsesin vitro and in vivo.

As used herein, the term “expression construct” or “transgene” isdefined as any type of genetic construct containing a nucleic acidcoding for gene products in which part or all of the nucleic acidencoding sequence is capable of being transcribed can be inserted intothe vector. The transcript is translated into a protein, but it need notbe. In certain embodiments, expression includes both transcription of agene and translation of mRNA into a gene product. In other embodiments,expression only includes transcription of the nucleic acid encodinggenes of interest.

As used herein, the term “expression vector” refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules or ribozymes. Expression vectors can contain avariety of control sequences, which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well and are described infra.

As used herein, the term “ex vivo” refers to “outside” the body. One ofskill in the art is aware that ex vivo and in vitro can be usedinterchangeably under certain circumstances.

The term “hyperproliferative disease” is defined as a disease thatresults from a hyperproliferation of cells. Exemplary hyperproliferativediseases include, but are not limited to cancer or autoimmune diseases.Other hyperproliferative diseases may include vascular occulsion,restenosis, atherosclerosis, or inflammatory bowel disease.

As used herein, the term “gene” is defined as a functional protein,polypeptide, or peptide-encoding unit. As will be understood by those inthe art, this functional term includes genomic sequences, cDNAsequences, and smaller engineered gene segments that express, or isadapted to express, proteins, polypeptides, domains, peptides, fusionproteins, and mutants.

The term “immunogenic composition” or “immunogen” refers to a substancethat is capable of provoking an immune response. Examples of immunogensinclude, e.g., antigens, autoantigens that play a role in induction ofautoimmune diseases, and tumor-associated antigens expressed on cancercells.

As used herein, the term “intracellular signaling domain” generallyrefers to a cytoplasmic tail of a membrane-associated receptor molecule,e.g., the cytoplasmic tail of CD40. The term also includes signalingmolecules like that in the zeta chain of CD3 or adaptor molecules thatengage down-stream cell signaling pathways.

As used herein, the term “multimerizing” or “multimerization” refers tothe complexation of three or more subunits into a single entity. Assuch, multimerization is distinct from “dimerizing” or “dimerization”which refers to the complexation of exactly two subunits into a singleentity.

As used herein, the term “pharmaceutically or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

As used herein, the term “polynucleotide” is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means. Furthermore, one skilled in the art iscognizant that polynucleotides include mutations of the polynucleotides,include but are not limited to, mutation of the nucleotides, ornucleosides by methods well known in the art.

As used herein, the term “polypeptide” is defined as a chain of aminoacid residues, usually having a defined sequence. As used herein theterm polypeptide is interchangeable with the terms “peptides” and“proteins.”

As used herein, the term “promoter” is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa gene.

As used herein, the term “self-adjuvanting” refers to a microbe orvector that carries with it both its own antigens and an adjuvantingmoiety. The microbe may be a virus, bacterium, fungus, parasite, orprotozoa. A vector construct may also be self-adjuvanting if it encodesboth an antigen or antigens and an adjuvanting moiety.

In the present invention, the term “therapeutic construct” may also beused to refer to the expression construct or transgene. One skilled inthe art realizes that the present invention utilizes the expressionconstruct or transgene as a therapy to treat hyperproliferative diseasesor disorders, such as cancer, thus the expression construct or transgeneis a therapeutic construct or a prophylactic construct.

The term “transfection” and “transduction” refer to the process by whichan exogenous DNA sequence is introduced into a eukaryotic host cell.Transfection is the non-viral delivery of nucleic acids (either DNA orRN) and can be achieved by any one of a number of means includingelectroporation, microinjection, gene gun delivery, retroviralinfection, lipofection, polymer-mediated delivery, and the like.Transfection refers to the delivery of nucleic acids by a virus or viralvector where the nucleic acids are typical DNA for a DNA virus and RNAfor an RNA virus. Also, for bacteria that enter cells such as Salmonellaor Listeria, plasmid DNA can be introduced into these bacteria whichthen carry that DNA into the eukaryotic host cell, a process called“bactofection.”

As used herein, the term “syngeneic” refers to cells, tissues or animalsthat have the same genotype. For example, identical twins or animals ofthe same inbred strain. Syngeneic and isogeneic can be usedinterchangeable.

The term “subject” as used herein includes, but is not limited to, anorganism or animal; a mammal, including, e.g., a human, non-humanprimate (e.g., monkey), mouse, pig, cow, goat, rabbit, rat, guinea pig,hamster, horse, monkey, sheep, or-other non-human mammal; a non-mammal,including, e.g., a non-mammalian vertebrate, such as a bird (e.g., achicken or duck) or a fish, and a non-mammalian invertebrate.

As used herein, the term “under transcriptional control” or “operativelylinked” is defined as the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene.

As used herein, the terms “treatment,” “treat,” “treated,” or “treating”refer to prophylaxis and/or therapy. When used with respect to aninfectious disease, for example, the term refers to a prophylactictreatment which increases the resistance of a subject to infection witha pathogen or, in other words, decreases the likelihood that the subjectwill become infected with the pathogen or will show signs of illnessattributable to the infection, as well as a treatment after the subjecthas become infected in order to fight the infection, e.g., reduce oreliminate the infection or prevent it from becoming worse.

As used herein, the term “vaccine” refers to a formulation whichcontains the composition of the present invention and which is in a faunthat is capable of being administered to an animal Typically, thevaccine includes a conventional saline or buffered aqueous solutionmedium in which the composition of the present invention is suspended ordissolved. In this form, a composition of the present invention can beused conveniently to prevent, ameliorate, or otherwise treat acondition. Upon introduction into a subject, the vaccine is able toprovoke an immune response including, but not limited to, the productionof antibodies, cytokines and/or other cellular responses.

A good example of the purposeful introduction of an adjuvant into avaccine was reported by Ruby et al. (1995) Nature Medicine 1: 437-441.These authors studied the vaccinia virus, a live viral vaccine thatnevertheless replicates very strongly in mice unless controlled by thehost response. In this case, the coding sequence for CD40 ligand (CD40L)was genetically introduced into recombinant vaccinia virus and tested inmice. When the vaccinia virus also led to the expression of CD40L, thereplication of the virus was severely curtained by host responses.

Another example was the report of Lin et al. (2009) J Virol 83:1216-1227. They found that incorporating CD40L onto the surface of asimian immunodeficiency virus (SIV) could lead to enhanced antibody andcellular immune responses. In this case, the SIV was further engineeredso that it could only undergo one round of infection (“single-cycle”virus), which ensured that it could not by itself lead to persistent SIVinfection.

CD40L is normally a membrane molecule. Consequently, the CD40L adjuvantmolecule appears on the surface of these enveloped (i.e.,membrane-bearing) viruses. To activate immune cells, these vaccines mustengage the extracellular portion of the CD40 receptor. The resultingCD40L-mediated stimulation of the CD40 receptor can occur on either thesame cells infected by the vaccinia-CD40L or SIV-CD40L constructs or onadjacent cells. The risk of activating an adjacent cell is that theadjacent cell might not be an antigen-presenting cell or, if it is anantigen-presenting cell, it might not have the proper antigen (e.g.,vaccinia antigens in the case of vaccinia-CD40L or SIV antigens in thecase of SIV-CD40L). Thus, expressing CD40L as a membrane protein on avirus or microbe does not ensure that the antigen and adjuvant areco-extensive in space and time.

As a way of solving this problem, gene therapy approaches has beendeveloped to target dendritic and activate them through the CD40pathway. The method relies on the cytoplasmic tale of CD40 whichcontains amino acid sequences that engage signal transduction molecules,chiefly the TNF-Receptor Activation Factors or TRAFs of which TRAF6 isone of the most important. The CD40 intracellular signaling domain canbe fused with a myristylation sequence that permitted the fusion proteinto attach itself to the inner leaflet of the plasma membrane. As afurther step, the fusion construct includes a sequence taken from theFK506 binding protein (FKBP12) that allowed the fusion protein to bedimerized by a drug composed of two FK506 moieties joined together(e.g., AP20187). The result is a chemically induced dimerization system(CID). When the entire genetic construct is expressed under the controlof the CD11c promoter that is preferentially activated in myeloiddendritic cells and introduced into these cells, then the entire systemmimics CD40 activation of dendritic cells once the chemical dimerizer(e.g., AP20187) is added. The drawbacks to this system include: (1) themyristylation-FK506 binding domain-CD40 cytoplasmic region fusionconstruct requires a genetic vector to introduce it into cells or elsestable transgenic cells must be produced; (2) the chemical dimerizermust be added and its pharmacology must be separately controlled; (3) ifthe expression of the activated CD40 signaling domain inside CD11cdendritic cells is considered to be an adjuvant, no method was providedto couple this system to an antigen such that the antigen and adjuvantare co-extensive in space and time; and (4) the description of the CIDsystem infers that it works by dimerization. The last point is importantbecause the intracellular domain of CD40 signals best when it is presentas a trimer, yet the description of Hanks et al. (2005) Nat Med 11:130-137, teaches away from this by implying that a dimer is sufficient.

Consequently, what is needed is a more general way to ensure that anantigen and adjuvant are co-extensive in space and time. In one aspect,this is performed without the need for a chemical crosslinker, e.g.,through a self-assembling intracellular complex. Using CD40 as anexample, such a self-assembling complex should lead to multimers ofthree or more CD40 intracellular cytoplasmic domains as an adjuvant andthis complex would need to be operatively linked to the antigen in orderthat the antigen and adjuvant be co-extensive in space and time.

Concerning a self-assembling intracellular complex that leads tosignaling through the CD40 pathway, this is naturally present in theEpstein-Barr virus (EBV) in the form of the latent membrane protein 1(LMP1). LMP1 has an N-terminus containing six transmembrane regions someof which interact with others to form a multimeric patch in the plane ofthe membrane. The C-terminal region of LMP1 contains TRAF-binding motifsthat signal in a similar manner to those from the CD40 intracellularregion.

Related to LMP1 is an artificial fusion protein containing themultimerizing, membrane-associated N-terminus of LMP1 conjoined with theintracytoplasmic domain of CD40, i.e., LMP1-CD40. Thismembrane-associated intracellular fusion protein mimics the constitutivesignaling of CD40 without the need for an external ligand.

LMP1 and LMP1-CD40 exemplify defined amino acid sequences that carrywith them the ability to activate the CD40 signaling pathway. CD40signaling is known to activate dendritic cells to present antigen to Tcells. CD40 signaling is known to activate B cells to present antigen toT cells. CD40 signaling is known to mature B cells intoantibody-producing cells. In particular, CD40 activated B cells undergo“class switching” of their antibody production from IgM to IgG or insome circumstances to IgA, and this can be replicated by LMP1.

However, prior to the present invention, it was not recognized that LMP1or LMP1-CD40 could be used as adjuvants and there was no suggestion asto how this might be accomplished. One of the central, non-obvious, andinventive aspects of the present invention is that it envisions LMP1 orLMP1-CD40 as portable adjuvant compositions that can be introduced intomicrobes, most preferably viruses, or tumor cells. These microbes ortumor cells carry their own antigens with them. By placing LMP1 orLMP1-CD40 directly into them, the antigen and adjuvant becomeco-extensive in space and time, which is an important feature of thepresent invention.

While the above discussion relates to the CD40 signaling pathway, theapplication of the present invention extends to other similar receptormolecules. These receptors are collectively known as the TNF ReceptorSuperFamily (TNFRSF). As a general rule, signaling by each and any ofthese receptors requires that their cytoplasmic domains be multimerizedinto complexes of three or more amino acid strands. Just as LMP1-CD40combines the multimerizing domain of LMP1 with the intracellularsignaling domain of the CD40 receptor into an amino acid sequencecassette, so too the present invention anticipates fusions of LMP1 withthe intracellular signaling domains of other TNFRSFs. Examples includeLMP1-4-1BB, LMP1-OX40, LMP1-CD27, LMP1-RANK, LMP1-BAFF-R, etc.Significantly, these other TNFRSFs are expressed on a different range ofcells than is CD40 and their stimulation has different effects. Forexample, the receptors for a proliferation-inducing ligand (APRIL)mediate cell growth such that their activation could be used to promotewound healing.

In the above discussion, note is made of fusing the LMP1 N-terminaldomain to the intracellular signaling domains of TNFRSFs such that thefusion protein self-assembles into a multimeric complex of three or morechains. While exemplary, the LMP1 N-terminal domain is not the onlyamino acid sequence that can be used in this manner.

The present invention provides compositions and methods which stimulatethe immune system to defend the host from microbes andhyperproliferative diseases including cancer. More particularly, thepresent invention provides compositions in which an amino acid sequenceof a multimerizing domain is fused to an intracytoplasmic signalingdomain that must be multimerized into a complex of three or moreidentical chains in order to provide an activating signal to a cell. Thecompositions of the present invention can be used as an adjuvant tobolster the immune response to a microbe or a tumor cell. In someembodiments, the composition of the present invention can be used tomake more effective preventative or therapeutic vaccines or to elicitthe immune control of tumors.

Certain embodiments of the present invention include an expressionconstruct comprising a polynucleotide promoter sequence, apolynucleotide sequence encoding the multimerization-intracellularsignaling fusion cassette, all operatively linked. It is envisioned thatthe expression construct is comprised within a vector forming anexpression vector; the vector is selected from the group consisting of aviral vector, a bacterial vector, and a mammalian vector. Themultimerization-intracellular signaling cassette can be comprised of anycombination of a multimerization domain with an intracellular signalingdomain where the multimerization domain leads to the complexation ofthree or more of the intracellular signaling domains.

Certain embodiments of the present invention are comprised of a virus orvector into which the multimerization-intracellular signaling cassettehas been inserted. There are at least two methods for determining wherein the virus or vector this cassette should be inserted: (1) if thecodons of a viral or vector gene can be deleted or mutated into anon-functional condition, then this location in the virus or vector is acandidate location for inserting the cassette. Most preferably, but notessentially, number of nucleic acid bases encoding the deleted ormutated viral or vector gene should be similar to the number of nucleicbases of the cassette being inserted (e.g., a dispensable viral orvector gene of about 400 bp can be replaced by cassette of 150 to 600bp); or (2) reports in the literature may already describe how foreigngenes can be inserted into a virus or vector. For example, enhancedgreen fluorescent protein (EGFP), beta-galactosidase, thymidine kinase,the neo gene for resistance to the toxicity of G418, beta-lactamase, andother enzymes have already been inserted into many viruses and vectorssuch that the viruses and vectors retain their functions of interest.Other examples of inserts that may already be described in theliterature include affinity tags for example calmodulin-binding peptide,cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase,FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag,SBP-tag, Strep-tag, and thioredoxin. Thus, those skilled in the art canreadily determine how to replace the enzyme or affinity tag insert witha multimerization-intracellular signaling cassette of the presentinvention.

Other embodiments of the present invention include a transduced cell, inwhich the cell is transduced with an expression vector containing themultimerization-intracellular signaling cassette. In one aspect, anantigen-presenting cell, for example a dendritic cell, can be loadedwith antigen and transduced with latent membrane protein 1 (LMP1) orLMP1-CD40 and then delivered to the host to initiate an immune response.

In another embodiment, a multimerization-intracellular signalingcassette can be transduced with an expression vector into a tumor cell.In another aspect where LMP1 or LMP1-CD40 is used, the tumor cellmembrane can carry the cassette. Since tumor cells are known to shedmicrovesicles that transfer membrane molecules including MHC Class Iwith antigen to other cells by a process known as trogocytosis ortransfer by small membrane fragments known as exosomes, this is amechanism whereby a tumor cell carrying themultimerization-intracellular signaling adjuvant cassette can donateantigen and adjuvant to antigen-presenting cells.

Another embodiment of the present invention is a pharmaceuticalcomposition including the virus or expression vector or a celltransduced with same, wherein the virus or expression vector leads tothe expression of the multimerization-intracellular signaling cassette.The virus or expression vector used for this pharmaceutical compositionincludes a polynucleotide promoter sequence, a first polynucleotidesequence encoding a multimerizing domain, and a second polynucleotidesequence encoding an intracellular signaling domain such that the seconddomain is in the correct reading frame with the multimerizing domain.These sequences may be followed by either a transcriptional terminationsignal and polyadenylation domain, or followed by an internal ribosomeentry sequence (IRES) followed by another separate coding sequence. Theresult is an operatively linked construct that leads to thetranscription and translation of the multimerization-intracellularsignaling cassette in a cell transfected by the pharmaceuticalcomposition.

As a further modification, a spacer or linker can be inserted betweenthe multimerization domain and the intracellular signaling domain of themultimerization-intracellular signaling cassette. Exemplary of suchspacers or linkers are nucleic acid sequences encoding the amino acidsequences GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGly-GlyGlySer (SEQ ID NO: 3),the hinge sequence from IgGGluProLysSerCysAspLysThrHisThrCys-ProProCysProAlaProGluLeuLeuGlyGlyPro(SEQ ID NO: 4), or one or more Gly or Ala residues. Without undue effortor experimentation, those skilled in the art can prepare variants of thebasic multimerization-intracellular signaling cassette containing suchlinkers and test them for their functional effects, choosing the mostefficacious construct for further application according to the presentinvention.

A further embodiment of the present invention includes a method formodulating an immune response in a subject. The method includes the stepof administering to the subject a virus or vector of the presentinvention. The virus or vector is expressed in an antigen-presentingcell, for example a dendritic cell or B cell. The virus or expressionvector used for this pharmaceutical composition includes apolynucleotide promoter sequence, a first polynucleotide sequenceencoding a multimerizing domain, and a second polynucleotide sequenceencoding an intracellular signaling domain such that the second domainis in the correct translational reading frame with the multimerizingdomain, all operatively linked.

Another embodiment includes a method of modulating an immune response ina subject. The method includes the steps of: transducing or transfectingan antigen-presenting cell with a virus or expression vector, whereinthe virus or expression vector includes a polynucleotide promotersequence, a polynucleotide sequence encoding themultimerizing-intracellular signaling cassette (with or without a spaceror linker), and a transcriptional terminator or IRES, all operativelylinked; and administering to the subject transduced antigen-presentingcells. Of note, if the method uses transduction of mRNA, no promotersequence is needed. In one aspect, the transduced or transfectedantigen-presenting cells enhance the immune response in the subject. Inanother aspect, the transduced or transfected antigen-presenting cellsare administered to the subject simultaneously to administration of anantigen or that the antigen is already encoded by the virus orexpression vector or already present in the host subject.

Another embodiment of the present invention is a method of inducing aregulated immune response against an antigen in a subject. The methodincludes the steps of: transducing or transfecting an antigen-presentingcell with a virus or expression vector, wherein the virus or expressionvector includes a polynucleotide promoter sequence, a polynucleotidesequence encoding the multimerizing-intracellular signaling cassette(with or without a spacer or linker), and a transcriptional terminatoror IRES, all operatively linked; loading transduced or transfectedantigen-presenting cells with the antigen; and administering transducedor transfected, loaded antigen-presenting cells to the subject therebyeffecting a cytotoxic T lymphocyte and natural killer cell anti-tumorantigen immune response. In one aspect, the transduced or transfected,loaded antigen-presenting cells are administered to the subjectintradermally, subcutaneously, intranodally or intralymphatically. Inanother aspect, the antigen-presenting cells are transduced ortransfected with the virus or expression vector in vitro or ex vivoprior to administering to the subject.

Loading the antigen-presenting cells with an antigen can be accomplishedutilizing standard methods, for example, pulsing, transducing,transfecting, and/or electrofusing. In one aspect, the antigen can benucleic acids (DNA or RNA), proteins, protein lysate, whole cell lysate,or antigen proteins linked to other proteins, e.g., heat shock proteins.

In various embodiments, the antigens can be derived or isolated from apathogenic microorganism for example viruses including Humanimmunodeficiency virus (HIV), influenza, Herpes simplex, human papillomavirus, Hepatitis B, Hepatitis C, EBV, Cytomegalovirus (CMV) and thelike. The antigen may be derived or isolated from pathogenic bacteriasuch as from Chlamydia, Mycobacteria, Legionella, Meningiococcus, GroupA Streptococcus, Salmonella, Listeria, Hemophilus influenzae, and thelike. In one aspect, the antigen may be derived or isolated frompathogenic yeast including Aspergillus, invasive Candida, Nocardia,Histoplasmosis, Cryptosporidia and the like. The antigen may be derivedor isolated from a pathogenic protozoan and pathogenic parasitesincluding, but not limited to Pneumocystis carinii, Trypanosoma,Leishmania, Plasmodium and Toxoplasma gondii.

In certain embodiments, the antigen includes an antigen associated witha preneoplastic or hyperplastic state. Antigens may also be associatedwith, or causative of cancer. In one aspect, such antigens include tumorspecific antigen, tumor associated antigen (TAA) or tissue specificantigen, epitope thereof, and epitope agonist thereof. In anotheraspect, such antigens include but are not limited to carcinoembryonicantigen (CEA) and epitopes thereof such as CAP-1, CAP-1-6D and the like,MART-1, MAGE-1, MAGE-3, GAGE, GP-100, MUC-1, MUC-2, point mutated rasoncogene, normal and point mutated p53 oncogenes, PSMA, tyrosinase,TRP-1 (gp75), NY-ESO-1, TRP-2, TAG72, KSA, CA-125, PSA,HER-2/neu/c-erb/B2, BRC-I, BRC-II, bcr-abl, pax3-fkhr, ews-fli-1,modifications of TAAs and tissue specific antigen, splice variants ofTAAs, epitope agonists, and the like.

Another embodiment is a method of treating and/or preventing a diseaseand/or disorder. The method includes administering to a subject aneffective amount of a virus or expression vector of the presentinvention to treat and/or prevent the disease and/or disorder, whereinthe expression vector includes a polynucleotide promoter sequence, apolynucleotide sequence encoding the multimerizing-intracellularsignaling cassette (with or without a spacer or linker), atranscriptional terminator or IRES, all operatively linked. Of note, ifthe method uses transduction of mRNA, no promoter sequence is needed. Anexemplary multimerizing-intracellular signaling cassette is LMP1 orLMP1-CD40.

In certain embodiments, the disease is a hyperproliferative disease,which can also be further defined as cancer. In still furtherembodiments, the cancer is melanoma, non-small cell lung, small-celllung, lung, hepatocarcinoma, leukemia, retinoblastoma, astrocytoma,glioblastoma, gum, tongue, neuroblastoma, head, neck, breast,pancreatic, prostate, renal, bone, testicular, ovarian, mesothelioma,cervical, gastrointestinal, lymphoma, brain, colon, sarcoma or bladder.The cancer may include a tumor comprised of tumor cells. For example,tumor cells may include, but are not limited to melanoma cell, a Maddercancer cell, a breast cancer cell, a lung cancer cell, a colon cancercell, a prostate cancer cell, a liver cancer cell, a pancreatic cancercell, a stomach cancer cell, a testicular cancer cell, a brain cancercell, an ovarian cancer cell, a lymphatic cancer cell, a skin cancercell, a brain cancer cell, a bone cancer cell, or a soft tissue cancercell.

In other embodiments, the hyperproliferative diseases include rheumatoidarthritis, inflammatory bowel disease, osteoarthritis, leiomyomas,adenomas, lipomas, hemangiomas, fibromas, vascular occlusion,restenosis, atherosclerosis, pre-neoplastic lesions (such as adenomatoushyperplasia and prostatic intraepithelial neoplasia), carcinoma in situ,oral hairy leukoplakia, psoriasis, or combinations thereof.

Yet further, another embodiment is a method of treating a disease and/ordisorder. The method includes administering to a subject an effectiveamount of a transduced or transfected antigen-presenting cell to treatthe disease and/or disorder, wherein the transduced or transfectedantigen-presenting cell is transduced or transfected with an expressionvector including a polynucleotide promoter sequence, a polynucleotidesequence encoding the multimerizing-intracellular signaling cassette(with or without a spacer or linker), and a transcriptional terminatoror IRES, all operatively linked. In one aspect, themultimerizing-intracellular signaling cassette includes amultimerization domain operatively linked to the intracellular signalingdomain of a TNFRSF, more specifically the CD40 cytoplasmic domain. Inanother aspect, the transduced or transfected antigen-presenting cellscan be administered to the subject intradermally, subcutaneously, orintranodally. The antigen-presenting cells can be transduced ortransfected with the virus or expression vector in vitro prior toadministering to the subject. The method may further includeelectrofusing the transduced or transfected antigen-presenting cell to atumor cell. In certain embodiments, the tumor cell is syngeneic, orallogeneic. The method may also further includes transfecting thetransduced or transfected antigen-presenting cell with tumor cell mRNAand/or pulsing the transduced or transfected antigen-presenting cellwith tumor cell protein lysates and/or pulsing the transduced ortransfected antigen-presenting cell with heat shock proteins linked totumor cell polypeptides.

Another embodiment is a method of treating a subject with cancer. Themethod includes administering to the patient an effective amount of atransduced or transfected antigen-presenting cell to treat the cancer.In one aspect, the transduced or transfected antigen-presenting cell istransduced with an expression vector or viral construct including apolynucleotide promoter sequence, a polynucleotide sequence encoding themultimerizing-intracellular signaling cassette (with or without a spaceror linker), and a transcriptional terminator or IRES, all operativelylinked; and administering at least one other anticancer treatment. Ofnote, if the method uses transduction of mRNA, no promoter sequence isneeded. In another aspect, the anticancer treatment is selected from thegroup consisting of chemotherapy, immunotherapy, surgery, radiotherapy,gene therapy and biotherapy.

In another embodiment, the multimerizing-intracellular signalingcassette (with or without a spacer or linker) can be delivered to atumor in vivo using a microbe or virus. Listeria is an example of abacterium that preferentially targets tumors and can transfer plasmidDNA to eukaryotic cells (“bactofection”). Oncolytic viruses are thosethat preferentially infect tumor cells in vivo. Examples of oncolyticviruses include adenoviruses, reoviruses, alphaviruses, Herpes Simplexvirus, Newcastle disease virus, Coxsackie B virus, Coxsackie A21 virus,Sindbis virus, measles virus, poliovirus, vesicular stomatitis virus,myxoma virus, vaccinia virus and other poxviruses, Sendai virus, andinfluenza virus. In this embodiment, the microbe or oncolytic viruscontaining the multimerizing-intracellular signaling cassette isinjected either intravenously, subcutaneously, or directly into thetumor or tumor bed. In one aspect, the composition is injected into thespace left after a tumor has been surgically removed, e.g., the space inthe brain tissue following surgical removal of glioblastoma.

Intracellular signaling domains of the TNF Receptor SuperFamily(TNFRSF): The official nomenclature of the TNFRSFs is maintained by theHuman Genome Organization (HUGO) at following URL that was accessed onMar. 22, 2010 at would wide web genenames.org/genefamily/tnfrsf.php.While definitive information on the human proteins follow below, theskilled artisan would be able to determine similar information onTNFRSFs from non-human mammals, birds, and fish, as well as allelicvariants of the reference sequences.

CD27 is GenBank Accession # M63928. It is UniProtKB/SwissProt reference# P26842, which gives its intracytoplasmic signaling domain as aminoacids 213-260.

CD40 is GenBank Accession # X60592. It is UniProtKB/SwissProt reference# P25942, which gives its intracytoplasmic signaling domain as aminoacids 216-277. However, for the constructs reported here, amino acids220-277 were used to provide an extra distance from the plasma membrane.

Fas is GenBank Accession # M67454. It is UniProtKB/SwissProt reference #P25445, which gives its intracytoplasmic signaling domain as amino acids191-335.

Lymphotoxin beta receptor (LTBR) is GenBank Accession # L04270. It isUniProtKB/SwissProt reference # P36941, which gives its intracytoplasmicsignaling domain as amino acids 239-435.

Nerve growth factor receptor (NGFR) is GenBank Accession # M14764. It isUniProtKB/SwissProt reference # P08138, which gives its intracytoplasmicsignaling domain as amino acids 273-427.

Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A) isGenBank Accession # M75866. It is UniProtKB/SwissProt reference #P19438, which gives its intracytoplasmic signaling domain as amino acids235-455.

Tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) isGenBank Accession # M32315. It is UniProtKB/SwissProt reference #P20333, which gives its intracytoplasmic signaling domain as amino acids288-461.

Tumor necrosis factor receptor superfamily member 4 (TNFRSF4 or OX40) isGenBank Accession # X75962. It is UniProtKB/SwissProt reference #P43489, which gives its intracytoplasmic signaling domain as amino acids236-277.

Tumor necrosis factor receptor superfamily member 8 (TNFRSF8 or CD30) isGenBank Accession # M83554. It is UniProtKB/SwissProt reference #P28908, which gives its intracytoplasmic signaling domain as amino acids408-595.

Tumor necrosis factor receptor superfamily member 9 (TNFRSF9, CD137, or4-1BB) is GenBank Accession # L12964. It is UniProtKB/SwissProtreference # Q07011, which gives its intracytoplasmic signaling domain asamino acids 214-255.

Tumor necrosis factor receptor superfamily member 10A (TNFRSF10A, DR4,Apo2, or TRAILR-1) is GenBank Accession # U90875. It isUniProtKB/SwissProt reference # O00220, which gives its intracytoplasmicsignaling domain as amino acids 263-468.

Tumor necrosis factor receptor superfamily member 10B (TNFRSF10B, DR5,or TRAIL-R2) is GenBank Accession # AF012628. It is UniProtKB/SwissProtreference # O14763, which gives its intracytoplasmic signaling domain asamino acids 232-440.

Tumor necrosis factor receptor superfamily member 10D (TNFRSF10D, R5, orTRAIL-R2) is GenBank Accession # AF029761. It is UniProtKB/SwissProtreference # Q9UBF6, which gives its intracytoplasmic signaling domain asamino acids 233-386.

Tumor necrosis factor receptor superfamily member 11A (TNFRSF11A orRANK) is GenBank Accession # AF018253. It is UniProtKB/SwissProtreference # Q9Y6Q6, which gives its intracytoplasmic signaling domain asamino acids 234-616.

Tumor necrosis factor receptor superfamily member 12A (TNFRSF12A, FN14,or TweakR) is GenBank Accession # AB035480. It is UniProtKB/SwissProtreference # Q9NP84, which gives its intracytoplasmic signaling domain asamino acids 102-129.

Tumor necrosis factor receptor superfamily member 13B (TNFRSF13B orTACI) is GenBank Accession # AF023614. It is UniProtKB/SwissProtreference # O14836, which gives its intracytoplasmic signaling domain asamino acids 187-293.

Tumor necrosis factor receptor superfamily member 13C (TNFRSF13C orBAFFR) is GenBank Accession # AF373846. It is UniProtKB/SwissProtreference # Q96RJ3, which gives its intracytoplasmic signaling domain asamino acids 100-184.

Tumor necrosis factor receptor superfamily member 14 (TNFRSF14, HVEM, orLIGHTR) is GenBank Accession # U70321. It is UniProtKB/SwissProtreference # Q92956, which gives its intracytoplasmic signaling domain asamino acids 224-283.

Tumor necrosis factor receptor superfamily member 17 (TNFRSF17 or BCMA)is GenBank Accession # Z29574. It is UniProtKB/SwissProt reference #Q02223, which gives its intracytoplasmic signaling domain as amino acids78-184.

Tumor necrosis factor receptor superfamily member 18 (TNFRSF18 or GITR)is GenBank Accession # AF125304. It is UniProtKB/SwissProt reference #Q9Y5U5, which gives its intracytoplasmic signaling domain as amino acids184-241.

Tumor necrosis factor receptor superfamily member 19 (TNFRSF19 or TROY)is GenBank Accession # AB040434. It is UniProtKB/SwissProt reference #Q9NS68, which gives its intracytoplasmic signaling domain as amino acids192-423.

Tumor necrosis factor receptor superfamily member 21 (TNFRSF21 or DR6)is GenBank Accession # AF068868. It is UniProtKB/SwissProt reference #Q9E8U5, which gives its intracytoplasmic signaling domain as amino acids371-655.

Tumor necrosis factor receptor superfamily member 25 (TNFRSF25 or DR3)is GenBank Accession # U72763. It is UniProtKB/SwissProt reference #Q93038, which gives its intracytoplasmic signaling domain as amino acids221-417.

Latent membrane protein 1 (LMP1) is not strictly a member of the TNFReceptor SuperFamily. However, we note here that the prototypic RAJIcell LMP1 is GenBank Accession # HS4LMP1. It is UniProtKB/SwissProtreference # P13198, which gives its intracytoplasmic signaling domain asamino acids 187-386.

Multimerizing domains: As described above, the prototypic multimerizingdomain for a multimerizing-intracellular signaling cassette is the LMP1protein of the RAJI strain of the Epstein-Barr Virus (EBV). It isGenBank Accession # HS4LMP1. It is UniProtKB/SwissProt reference #P13198, which gives its N-terminal domain containing 6 transmembraneregions as amino acids 1-186. However, to provide distance from theinner leaflet of the plasma membrane, amino acids 1-190 were used (4extra amino acids retained).

Design principles of a multimerizing-intracellular signaling cassette:For many members of the TNF Receptor SuperFamily (TNFRSF), clustering inthe plane of the cell membrane is required to generate an intracellularsignaling event. In order to produce a constitutively activated TNFRSF,it is necessary to mimic this clustering by fusing the intracytoplasmicdomain of the TNFRSF with a multimerizing motif. As noted above, theN-terminus of LMP1 is a very suitable multimerizing domain thateffectively activates either its own intracytoplasmic (=intracellular)domain (which has activities in common with that of CD40) or theheterologous intracytoplasmic domain of CD40. As described herein, LMP1is a complete multimerizing-intracellular signaling domain in and ofitself. LMP1-CD40 refers to the fusion of RAJI EBV LMP1 amino acids1-186 in frame with the intracytoplasmic domain of CD40 amino acids216-277.

As noted above, a spacer or linker can be inserted between themultimerizing domain and the intracellular signaling domain.

By analogy with LMP1-CD40, we predict that the fusion of the LMP1N-terminus with the intracytoplasmic signaling domains of each and anyof the TNFRSFs listed above will yield useful cassettes.

Insertion of a multimerizing-intracellular signaling cassette into HIV-1and other lentiviruses: a design prototype: As described above, the nefreading frame is ideal for the insertion of LMP1 or LMP1-CD40. Numerousproviral constructs have been made in which novel coding regions havebeen inserted into the nef region. Importantly, HIV-1 can replicate ifEGFP is placed in the nef-spliced message followed by an IRES directingthe translation of NEF protein. This sets a design precedent that is theprototype for the present invention: if a virus or virus vector has aregion that can be deleted entirely like nef or has been used to expressa heterologous coding sequence (e.g., EGFP or other markers describedabove), then this site is likely to be suitable for replacement by amultimerizing-intracytoplasmic signaling cassette. Performing thisreplacement will yield a virus with new properties depending upon thecells infected by the virus and the nature of the intracytoplasmicsignaling domain.

Lentiviruses have also been used as the basis for gene therapy. Oneexample is the use of lentiviruses to transduce dendritic cells for usein vaccines. From the description therein, one skilled in the art willknow how to insert a multimerizing-intracellular signaling cassette suchas LMP1 or LMP1-CD40 in order to improve the strength of this DC-basedvaccine strategy.

Insertion of a multimerizing-intracellular signaling cassette intopoxviruses: A prototypic poxvirus is Modified Vaccinia Ankara (MVA)which has been chosen as a vaccine to prevent small pox and as a vaccinevector. Other viruses in the poxvirus family that have been promoted foruse in humans include vaccinia (dryvax), NYVAC, and avipox (ALVAC). Tointroduce a multimerizing-intracellular signaling cassette into MVA,tools involving cloning LMP1 into the transfer vector, pLW-44, can beused (Wyatt L S, Shors S T, Murphy B R, Moss B. 1996. Development of areplication-deficient recombinant vaccinia virus vaccine effectiveagainst parainfluenza virus 3 infection in an animal model. Vaccine 14:1451-8). This transfer vector is a plasmid that contains greenfluorescent protein (GFP) and space for a second gene, here LMP1.Flanking regions homologous to MVA permit recombination of the constructinto the del III region of MVA. Recombinant viruses are generated inchicken embryo fibroblasts (CEF) and selected by EGFP fluorescence orimmunostaining with anti-EGFP antibody.

Insertion of a multimerizing-intracellular signaling cassette into anoncolytic virus: Viruses that target tumors are being studied for theiranti-tumor effects. Typically, these “oncolytic” viruses lead to tumorcell death. One exemplary oncolytic virus is measles virus. For example,use of Edmonton-NSe vaccine strain has been developed, by displayinghuman IL-13 at the C-terminus of the H protein, and introducing CD46 andsignaling lymphocyte activation molecule (SLAM)-ablating mutations in H.The skilled artisan would readily know how to replace the IL-13 codingsequence in this virus with LMP1, LMP1-CD40, or anothermultimerizing-intracellular signaling cassette. For example, LMP1-Fas,LMP1-TRAIL-R1, LMP1-TRAIL-R2, and LMP1-TNF-R1 are all examples where thecytoplasmic tail of the listed receptors can be multimerized by the LMP1N-terminal domain. The present invention provides that such engineeredviruses will be highly cytolytic for tumor cells and therefore usefulfor local therapy, e.g., treatment of brain tumors. Likewise, LMP1,LMP1-CD40, LMP1-4-1BB, LMP1-CD27, LMP1-OX40, LMP1-RANK, or LMP1-BAFFRcould be similarly introduced into measles virus and used to augment theimmune response to tumors. A similar approach could be taken using anyof the oncolytic viruses well known in the art.

Various oncolytic viruses have been disclosed in U.S. Pat. Nos.7,811,582, 7,731,952, 7,638,318, 7,595,042, 7,537,924, 7,459,154,7,267,815, 7,262,033, 7,223,593, 7,122,182, 7,118,755, 7,063,851,7,063,835, 7,030,099, 6,821,753, 6,719,982, 6,713,067, 6,641,817,6,544,770, 6,428,968, and 6,316,185; the contents of which areincorporated by reference in their entireties. Additional oncolyticviruses have also been disclosed in U.S. Patent Publication Nos.2009/0220460, 2007/0264282, 2006/0188480, 2006/0121522, 2005/0249707,2004/0219167, 2004/0063094, 2004/0022812, and 2003/0219409; the contentsof which are incorporated by reference in their entireties. The presentinvention provides that these oncolytic viruses can be modified toinclude an expression cassette for expressing the protein of theinvention, for example, LPM1 or LMP1-CD40, to stimulate immuneresponses. The present invention provides that the modified oncolyticviruses can be used in various vaccine compositions.

The present invention provides that DCs can be transfected ex vivo withmRNA encoding LMP1 or LMP1-CD40. These DCs can become activated to bebetter antigen-presenting cells. These antigen-pulsed DCs in culture canthen be administered to a subject, similar to the Dendreon Provenge™vaccine for prostate cancer.

The present invention also provides the use of plasmids expressing LMP1(pLMP1) or LMP1-CD40 (pMLP1-CD40) as DNA vaccine adjuvants. Such DNAvaccine can be used, for example, to prevent melanoma metastases.

The present invention also provides the use ofmultimerizing-intracellular signaling gene cassettes where the signalingportion is not from CD40. For example, some TNF receptor superfamilymembers (shown in FIG. 29) can be used to mediate growth. A LMP1 version(fusion protein) with any of these TNF receptor superfamily members canbe used for various indications, for example would healing and/orregenerative medicine.

Cis- versus Trans- effects of a multimerizing-intracellular signalingcassette: The expression of LMP1 in cells has been shown to inducepatched of the cell membrane to bleb off as microvesicles calledexosomes. LMP1-induced exosomes can then fuse with nearby cells bringingwith them both LMP1 and cellular RNAs (Meckes D G, Jr., Shair K H,Marquitz A R, Kung C P, Edwards R H, Raab-Traub N. 2010. Human tumorvirus utilizes exosomes for intercellular communication. Proc Natl AcadSci USA 107: 20370-5). This process is a variation on a phenomenon knownas “trogocytosis.” Alternative forms of trogocytosis have been reported.By this mechanism, tumor cells transduced or transfected to express LMP1or the multimerizing-intracellular signaling cassette can produceexosomes that are then taken up by antigen-presenting cells. It ispredicted that this will both activate such antigen-presenting cells(e.g., dendritic cells) and cause them to present tumor cell-derivedantigens to the immune system. This is called a trans effect because itaffects cells other than those in which the multimerizing-intracellularsignaling cassette was introduced by transfection or transduction. Thisallows the introduction of a multimerizing-intracellular signalingcassette to have more widespread effects than those due to just thecells that originally receive the construct (which is said to act in cisin such cells). In particular, a multimerizing-intracellular signalingcassette carried into a tumor cell by an oncolytic virus or microbe ispredicted to induce the formation of exosomes that are taken up by DCsand other antigen-presenting cells, leading to a stronger antitumorimmune response than would otherwise occur. This in turn will increasethe therapeutic effectiveness of oncolytic viruses or microbes.

Methods of Gene Transfer: In order to mediate the effect of thetransgene expression in a cell, it will be necessary to transfer theexpression constructs of the present invention into a cell. Suchtransfer may employ viral or non-viral methods of gene transfer. Thissection provides a discussion of methods and compositions of genetransfer.

A transformed cell including an expression vector is generated byintroducing into the cell the expression vector. Suitable methods forpolynucleotide delivery for transformation of an organelle, a cell, atissue or an organism for use with the current invention includevirtually any method by which a polynucleotide (e.g., DNA or RNA) can beintroduced into an organelle, a cell, a tissue or an organism, asdescribed herein or as would be known to one of ordinary skill in theart.

A host cell can, and has been, used as a recipient for vectors. Hostcells may be derived from prokaryotes or eukaryotes, depending uponwhether the desired result is replication of the vector or expression ofpart or all of the vector-encoded polynucleotide sequences. Numerouscell lines and cultures are available for use as a host cell, and theycan be obtained through the American Type Culture Collection (ATCC),which is an organization that serves as an archive for living culturesand genetic materials. In specific embodiments, the host cell is adendritic cell, which is an antigen-presenting cell.

It is well within the knowledge and skill of a skilled artisan todetermine an appropriate host. Generally this is based on the vectorbackbone and the desired result. A plasmid or cosmid, for example, canbe introduced into a prokaryote host cell for replication of manyvectors. Bacterial cells used as host cells for vector replicationand/or expression include DH5α, JM109, and KC8, as well as a number ofcommercially available bacterial hosts such as SURE™. Competent Cellsand SOLOPACK™ Gold Cells (Stratagene, La Jolla, Calif.). Alternatively,bacterial cells such as E. colt LE392 could be used as host cells forphage viruses. Eukaryotic cells that can be used as host cells include,but are not limited to yeast, insects and mammals. Examples of mammalianeukaryotic host cells for replication and/or expression of a vectorinclude, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO,Saos, and PC12. Examples of yeast strains include, but are not limitedto, YPH499, YPH500 and YPH501.

Non-Viral Transfer - Ex vivo Transformation: Methods for transfectingcells and tissues removed from an organism in an ex vivo setting areknown to those of skill in the art. For example, canine endothelialcells have been genetically altered by retroviral gene transfer in vitroand transplanted into a canine. In another example, Yucatan minipigendothelial cells were transfected by retrovirus in vitro andtransplanted into an artery using a double-balloon catheter. Thus, it iscontemplated that cells or tissues may be removed and transfected exvivo using the polynucleotides of the present invention. In particularaspects, the transplanted cells or tissues may be placed into anorganism. Thus, it is well within the knowledge of one skilled in theart to isolate dendritic cells from an animal, transfect the cells withthe expression vector and then administer the transfected or transformedcells back to the animal.

Injection: In certain embodiments, a polynucleotide may be delivered toan organelle, a cell, a tissue or an organism via one or more injections(i.e., a needle injection), such as, for example, subcutaneously,intradermally, intramuscularly, intravenously, intraperitoneally, etc.Methods of injection of vaccines are well known to those of ordinaryskill in the art (e.g., injection of a composition comprising a salinesolution). Further embodiments of the present invention include theintroduction of a polynucleotide by direct microinjection. The amount ofthe expression vector used may vary upon the nature of the antigen aswell as the organelle, cell, tissue or organism used.

Intradermal, intranodal, or intralymphatic injections are some of themore commonly used methods of DC administration. Intradermal injectionis characterized by a low rate of absorption into the bloodstream butrapid uptake into the lymphatic system. The presence of large numbers ofLangerhans dendritic cells in the dermis will transport intact as wellas processed antigen to draining lymph nodes. Proper site preparation isnecessary to perform this correctly (i.e., hair must be clipped in orderto observe proper needle placement). Intranodal injection allows fordirect delivery of antigen to lymphoid tissues. Intralymphatic injectionallows direct administration of DCs.

Electroporation: In certain embodiments of the present invention, apolynucleotide is introduced into an organelle, a cell, a tissue or anorganism via electroporation. Electroporation involves the exposure of asuspension of cells and DNA to a high-voltage electric discharge. Insome variants of this method, certain cell wall-degrading enzymes, suchas pectin-degrading enzymes, are employed to render the target recipientcells more susceptible to transformation by electroporation thanuntreated cells (U.S. Pat. No. 5,384,253, incorporated herein byreference).

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes, and rat hepatocytes have been transfectedwith the chloramphenicol acetyltransferase gene.

Calcium Phosphate: In other embodiments of the present invention, apolynucleotide is introduced to the cells using calcium phosphateprecipitation. Human KB cells have been transfected with adenovirus 5DNA. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK,NIH3T3 and HeLa cells were transfected with a neomycin marker gene, andrat hepatocytes were transfected with a variety of marker genes.

DEAE-Dextran: In another embodiment, a polynucleotide is delivered intoa cell using DEAE-dextran followed by polyethylene glycol. In thismanner, reporter plasmids were introduced into mouse myeloma anderythroleukemia cells.

Sonication Loading: Additional embodiments of the present inventioninclude the introduction of a polynucleotide by direct sonic loading.LTK-fibroblasts have been transfected with the thymidine kinase gene bysonication loading.

Liposome-Mediated Transfection: In a further embodiment of theinvention, a polynucleotide may be entrapped in a lipid complex, forexample, a liposome. Liposomes are vesicular structures characterized bya phospholipid bilayer membrane and an inner aqueous medium.Multilamellar liposomes have multiple lipid layers separated by aqueousmedium. They form spontaneously when phospholipids are suspended in anexcess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers. Alsocontemplated is a polynucleotide complexed with Lipofectamine (GibcoBRL) or Superfect (Qiagen).

Receptor Mediated Transfection: Still further, a polynucleotide may bedelivered to a target cell via receptor-mediated delivery vehicles.These take advantage of the selective uptake of macromolecules byreceptor-mediated endocytosis that will be occurring in a target cell.In view of the cell type-specific distribution of various receptors,this delivery method adds another degree of specificity to the presentinvention.

Certain receptor-mediated gene targeting vehicles include a cellreceptor-specific ligand and a polynucleotide-binding agent. Anotherembodiment includes a cell receptor-specific ligand to which thepolynucleotide to be delivered has been operatively attached. Severalligands have been used for receptor-mediated gene transfer (EP 0273085),which establishes the operability of the technique. Specific delivery inthe context of another mammalian cell type has been described. Incertain aspects of the present invention, a ligand is chosen tocorrespond to a receptor specifically expressed on the target cellpopulation.

In other embodiments, a polynucleotide delivery vehicle component of acell-specific polynucleotide-targeting vehicle may include a specificbinding ligand in combination with a liposome. The polynucleotide(s) tobe delivered can be housed within the liposome and the specific bindingligand can be functionally incorporated into the liposome membrane. Theliposome will thus specifically bind to the receptor(s) of a target celland deliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor-mediated delivery of a polynucleotide tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the polynucleotide delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichcan include one or more lipids or glycoproteins that directcell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialoganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes. The present invention provides that the tissue-specifictransforming constructs of the present invention can be specificallydelivered into a target cell in a similar manner.

Microprojectile Bombardment: Microprojectile bombardment techniques canbe used to introduce a polynucleotide into at least one, organelle,cell, tissue or organism (U.S. Pat. No. 5,550,318; U.S. Pat. No.5,538,880; U.S. Pat. No. 5,610,042; and PCT Application WO 94/09699;each of which is incorporated herein by reference). This method dependson the ability to accelerate DNA-coated microprojectiles to a highvelocity allowing them to pierce cell membranes and enter cells withoutkilling them. There are a wide variety of microprojectile bombardmenttechniques known in the art, many of which are applicable to theinvention.

In this microprojectile bombardment, one or more particles may be coatedwith at least one polynucleotide and delivered into cells by apropelling force. Several devices for accelerating small particles havebeen developed. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force.The microprojectiles used have consisted of biologically inertsubstances for example tungsten or gold particles or beads. Exemplaryparticles include those comprised of tungsten, platinum, and preferably,gold. The present invention provides that in some embodiments DNAprecipitation onto metal particles would not be necessary for DNAdelivery to a recipient cell using microprojectile bombardment. However,the present invention provides that particles may contain DNA ratherthan be coated with DNA. DNA-coated particles may increase the level ofDNA delivery via particle bombardment but are not, in and of themselves,necessary.

Viral Vector-Mediated Transfer: In certain embodiments, transgene isincorporated into a viral particle to mediate gene transfer to a cell.Typically, the virus simply will be exposed to the appropriate host cellunder physiologic conditions, permitting uptake of the virus. Thepresent methods can be advantageously employed using a variety of viralvectors.

Adenovirus: Adenovirus is particularly suitable for use as a genetransfer vector because of its mid-sized DNA genome, ease ofmanipulation, high titer, wide target-cell range, and high infectivity.The roughly 36 kb viral genome is bounded by 100-200 base pair (bp)inverted terminal repeats (ITR), in which are contained cis-actingelements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome that contain differenttranscription units are divided by the onset of viral DNA replication.

The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff. The products of the late genes (L1, L2, L3, L4 and L5), includingthe majority of the viral capsid proteins, are expressed only aftersignificant processing of a single primary transcript issued by themajor late promoter (MLP). The MLP (located at 16.8 map units) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence, which makes them preferred mRNAs for translation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.The two goals are, to an extent, coterminous, in that elimination ofadenoviral genes serves both ends. By practice of the present invention,it is possible achieve both these goals while retaining the ability tomanipulate the therapeutic constructs with-relative ease.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100-200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus. Therefore, inclusion of these elements in anadenoviral vector can permit replication.

In addition, the packaging signal for viral encapsulation is localizedbetween 194-385 bp (0.5-1.1 map units) at the left end of the viralgenome. This signal mimics the protein recognition site in bacteriophagelamda DNA where a specific sequence close to the left end, but outsidethe cohesive end sequence, mediates the binding to proteins that arerequired for insertion of the DNA into the head structure. E1substitution vectors of Ad have demonstrated that a 450 bp (0-1.25 mapunits) fragment at the left end of the viral genome could directpackaging in 293 cells.

It has been shown that certain regions of the adenoviral genome can beincorporated into the genome of mammalian cells and the genes encodedthereby expressed. These cell lines are capable of supporting thereplication of an adenoviral vector that is deficient in the adenoviralfunction encoded by the cell line. There also have been reports ofcomplementation of replication deficient adenoviral vectors by “helping”vectors, e.g., wild-type virus or conditionally defective mutants.

Replication-deficient adenoviral vectors can be complemented, in trans,by helper virus. This observation alone does not permit isolation of thereplication-deficient vectors, however, since the presence of helpervirus, needed to provide replicative functions, would contaminate anypreparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element, as provided for in thepresent invention, derives from the packaging function of adenovirus.

It has been shown that a packaging signal for adenovirus exists in theleft end of the conventional adenovirus map. Later studies showed that amutant with a deletion in the E1A (194-358 bp) region of the genome grewpoorly even in a cell line that complemented the early (E1A) function.When a compensating adenoviral DNA (0-353 bp) was recombined into theright end of the mutant, the virus was packaged normally. Furthermutational analysis identified a short, repeated, position-dependentelement in the left end of the adenovirus type 5 (Ad5) genome. One copyof the repeat was found to be sufficient for efficient packaging ifpresent at either end of the genome, but not when moved towards theinterior of the Ad5 DNA molecule.

By using mutated versions of the packaging signal, it is possible tocreate helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals is packagedselectively when compared to the helpers. If the preference is greatenough, stocks approaching homogeneity can be achieved.

Retrovirus: The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription. The resultingDNA then stably integrates into cellular chromosomes as a provirus anddirects synthesis of viral proteins. The integration results in theretention of the viral gene sequences in the recipient cell and itsdescendants. The retroviral genome contains at least three genes-gag,pol and env-that code for capsid proteins, polymerase enzyme, andenvelope components, respectively. A sequence found upstream from thegag gene, termed PSI (Ψ), functions as a signal for packaging of thegenome into virions. Two long terminal repeat (LTR) sequences arepresent at the 5′ and 3′ ends of the viral genome. These contain strongpromoter and enhancer sequences and also are required for integration inthe host cell genome.

In order to construct a retroviral vector, a nucleic acid encoding apromoter is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol and envgenes but without the LTR and PSI (Ψ) components is constructed. When arecombinant plasmid containing a human cDNA, together with theretroviral LTR and PSI (Ψ) sequences is introduced into this cell line(by calcium phosphate precipitation for example), the PSI (Ψ) sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media. Themedia containing the recombinant retroviruses is collected, optionallyconcentrated, and used for gene transfer. Retroviral vectors are able toinfect a broad variety of cell types. However, integration and stableexpression of many types of retroviruses require the division of hostcells.

An approach designed to allow specific targeting of retrovirus vectorshas been developed based on the chemical modification of a retrovirus bythe chemical addition of galactose residues to the viral envelope. Thismodification can permit the specific infection of cells such ashepatocytes via asialoglycoprotein receptors.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodiescan be coupled via the biotin components by using streptavidin. Usingantibodies against major histocompatibility complex class I and class IIantigens, the infection of a variety of human cells that bore thosesurface antigens has been demonstrated with an ecotropic virus in vitro.

Adeno-Associated Virus (AAV) utilizes a linear, single-stranded DNA ofabout 4700 base pairs. Inverted terminal repeats flank the genome. Twogenes are present within the genome, giving rise to a number of distinctgene products. The first, the cap gene, produces three different virionproteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene,encodes four non-structural proteins (NS). One or more of these rep geneproducts is responsible for transactivating AAV transcription.

The three promoters in AAV are designated by their location, in mapunits, in the genome. These are, from left to right, p5, p19 and p40.Transcription gives rise to six transcripts, two initiated at each ofthree promoters, with one of each pair being spliced. The splice site,derived from map units 42-46, is the same for each transcript. The fournon-structural proteins apparently are derived from the longer of thetranscripts, and three virion proteins all arise from the smallesttranscript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The mostwell-characterized helpers can be adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low-levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

The terminal repeats of the AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as p201, which containsa modified AAV genome, or by other methods known to the skilled artisan,including but not limited to chemical or enzymatic synthesis of theterminal repeats based upon the. published sequence of AAV. Theordinarily skilled artisan can determine, by well-known methods such asdeletion analysis, the minimum sequence or part of the AAV ITRs which isrequired to allow function, i.e., stable and site-specific integration.The ordinarily skilled artisan also can determine which minormodifications of the sequence can be tolerated while maintaining theability of the terminal repeats to direct stable, site-specificintegration.

AAV-based vectors have proven to be safe and effective vehicles for genedelivery in vitro, and these vectors are being developed and tested inpre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo.

AAV-mediated efficient gene transfer and expression in the lung has ledto clinical trials for the treatment of cystic fibrosis. Similarly, theprospects for treatment of muscular dystrophy by AAV-mediated genedelivery of the dystrophin gene to skeletal muscle, of Parkinson'sdisease by tyrosine hydroxylase gene delivery to the brain, ofhemophilia B by Factor IX gene delivery to the liver, and potentially ofmyocardial infarction by vascular endothelial growth factor gene to theheart, appear promising since AAV-mediated transgene expression in theseorgans has recently been shown to be highly efficient.

Other Viral Vectors: Other viral vectors are employed as expressionconstructs in the present invention. Vectors derived from viruses suchas vaccinia virus canary pox virus, and herpes viruses are employed.These viruses offer several features for use in gene transfer intovarious mammalian cells.

Once the construct has been delivered into the cell, the nucleic acidencoding the transgene are positioned and expressed at different sites.In certain embodiments, the nucleic acid encoding the transgene isstably integrated into the genome of the cell. This integration is inthe cognate location and orientation via homologous recombination (genereplacement) or it is integrated in a random, non-specific location(gene augmentation). In yet further embodiments, the nucleic acid isstably maintained in the cell as a separate, episomal segment of DNA.Such nucleic acid segments or “episomes” encode sequences sufficient topermit maintenance and replication independent of or in synchronizationwith the host cell cycle. How the expression construct is delivered to acell and where in the cell the nucleic acid remains is dependent on thetype of expression construct employed.

Formulations and Routes for Administration to Patients: Where clinicalapplications are contemplated, it will be necessary to preparepharmaceutical compositions-expression constructs, expression vectors,fused proteins, transduced or transfected cells, activated DCs,transduced or transfected and loaded DCs-in a form appropriate for theintended application. Generally, this will entail preparing compositionsthat are essentially free of pyrogens, as well as other impurities thatcould be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present invention include aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. A pharmaceuticallyacceptable carrier includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like. The use of such media and agents forpharmaceutically active substances is well know in the art. Exceptinsofar as any conventional media or agent is incompatible with thevectors or cells of the present invention, its use in therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

For oral administration, the compositions of the present invention maybe incorporated with excipients and used in the form of non-ingestiblemouthwashes and dentifrices. A mouthwash may be prepared incorporatingthe active ingredient in the required amount in an appropriate solvent,such as a sodium borate solution (Dobell's Solution). Alternatively, theactive ingredient may be incorporated into an antiseptic wash containingsodium borate, glycerin and potassium bicarbonate. The active ingredientalso may be dispersed in dentifrices, including: gels, pastes, powdersand slurries. The active ingredient may be added in a therapeuticallyeffective amount to a paste dentifrice that may include water, binders,abrasives, flavoring agents, foaming agents, and humectants.

The compositions of the present invention may be formulated in a neutralor salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media, which can be employed, will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, Remington's PharmaceuticalSciences 15th Edition, pages 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and general safety and purity standards as required by FDAOffice of Biologics standards.

Methods for Treating a Disease: The present invention also encompassesmethods of treatment or prevention of a disease caused by pathogenicmicroorganisms and/or a hyperproliferative disease.

Diseases may be treated or prevented by use of the present inventioninclude diseases caused by viruses, bacteria, yeast, parasites,protozoa, cancer cells and the like. The pharmaceutical composition ofthe present invention (transduced or transfected DCs, expression vector,expression construct, etc.) of the present invention may be used as ageneralized immune enhancer (DC activating composition or system) and assuch has utility in treating diseases. Exemplary diseases that can betreated and/or prevented utilizing the pharmaceutical composition of thepresent invention include, but are not limited to infections of viraletiology such as HIV, influenza, Herpes, viral hepatitis, Epstein Barr,polio, viral encephalitis, measles, chicken pox, Papilloma virus etc.;or infections of bacterial etiology such as pneumonia, tuberculosis,syphilis, etc.; or infections of parasitic etiology such as malaria,trypanosomiasis, leishmaniasis, trichomoniasis, amoebiasis, etc.

Preneoplastic or hyperplastic states which may be treated or preventedusing the pharmaceutical composition of the present invention(transduced or transfected DCs, expression vector, expression construct,etc.) of the present invention include but are not limited topreneoplastic or hyperplastic states such as colon polyps, Crohn'sdisease, ulcerative colitis, breast lesions and the like.

Cancers which may be treated using the pharmaceutical composition of thepresent invention of the present invention include, but are not limitedto primary or metastatic melanoma, adenocarcinoma, squamous cellcarcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, sarcoma,lung cancer, liver cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma,leukemias, uterine cancer, breast cancer, prostate cancer, ovariancancer, pancreatic cancer, colon cancer, multiple myeloma,neuroblastoma, NPC, bladder cancer, cervical cancer and the like.

Other hyperproliferative diseases that may be treated using DCactivation system of the present invention include, but are not limitedto rheumatoid arthritis, inflammatory bowel disease, osteoarthritis,leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascularocclusion, restenosis, atherosclerosis, pre-neoplastic lesions (such asadenomatous hyperplasia and prostatic intraepithelial neoplasia),carcinoma in situ, oral hairy leukoplakia, or psoriasis.

In the method of treatment, the administration of the pharmaceuticalcomposition (expression construct, expression vector, fused protein,transduced or transfected cells, activated DCs, transduced ortransfected and loaded DCs) of the invention may be for either“prophylactic” or “therapeutic” purpose. When provided prophylactically,the pharmaceutical composition of the present invention is provided inadvance of any symptom. The prophylactic administration ofpharmaceutical composition serves to prevent or ameliorate anysubsequent infection or disease. When provided therapeutically, thepharmaceutical composition is provided at or after the onset of asymptom of infection or disease. Thus the present invention may beprovided either prior to the anticipated exposure to a disease-causingagent or disease state or after the initiation of the infection ordisease.

The term “unit dose” as it pertains to the inoculum refers to physicallydiscrete units suitable as unitary dosages for mammals, each unitcontaining a predetermined quantity of pharmaceutical compositioncalculated to produce the desired immunogenic effect in association withthe required diluent. The specifications for the novel unit dose of aninoculum of this invention are dictated by and are dependent upon theunique characteristics of the pharmaceutical composition and theparticular immunologic effect to be achieved.

An effective amount of the pharmaceutical composition would be theamount that achieves this selected result of enhancing the immuneresponse, and such an amount could be determined as a matter of routineby a person skilled in the art. For example, an effective amount of fortreating an immune system deficiency could be that amount necessary tocause activation of the immune system, resulting in the development ofan antigen specific immune response upon exposure to antigen. The termis also synonymous with “sufficient amount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One of ordinary skillin the art can empirically determine the effective amount of aparticular composition of the present invention without necessitatingundue experimentation.

Genetic Based Therapies: Specifically, the present inventors intend toprovide, to a cell, an expression construct capable of providing aco-stimulatory polypeptide, such as LMP1 or LMP1-CD40 to the cell, suchas an antigen-presenting cell. The lengthy discussion of expressionvectors and the genetic elements employed therein is incorporated intothis section by reference. Particularly preferred expression vectors areviral vectors such as adenovirus, adeno-associated virus, herpes virus,vaccinia virus and retrovirus. Also preferred is lysosomal-encapsulatedexpression vector.

Those of skill in the art are well aware of how to apply gene deliveryto in vivo and ex vivo situations. For viral vectors, one generally willprepare a viral vector stock. Depending on the kind of virus and thetiter attainable, one will deliver 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸,1×10⁹, 1×10¹⁰, 1×10¹¹ or 1×10¹² infectious particles to the patient.Similar figures may be extrapolated for liposomal or other non-viralformulations by comparing relative uptake efficiencies. Formulation as apharmaceutically acceptable composition is discussed below.

Cell Based Therapy: Another therapy that is contemplated is theadministration of transduced or transfected antigen-presenting cells.The antigen-presenting cells may be transduced or transfected in vitro.Formulation as a pharmaceutically acceptable composition is discussedabove.

In cell based therapies, the transduced or transfectedantigen-presenting cells may be transduced or transfected with targetantigen nucleic acids, such as mRNA or DNA or proteins; pulsed with celllysates, proteins or nucleic acids; or electrofused with cells. Thecells, proteins, cell lysates, or nucleic acid may derive from cells,such as tumor cells or other pathogenic microorganism, for example,viruses, bacteria, protozoa, etc.

Combination Therapies: In order to increase the effectiveness of theexpression vector of the present invention, it may be desirable tocombine these compositions and methods of the invention with an agenteffective in the treatment of the disease.

In certain embodiments, anti-cancer agents may be used in combinationwith the present invention. An “anti-cancer” agent is capable ofnegatively affecting cancer in a subject, for example, by killing one ormore cancer cells, inducing apoptosis in one or more cancer cells,reducing the growth rate of one or more cancer cells, reducing theincidence or number of metastases, reducing a tumor's size, inhibiting atumor's growth, reducing the blood supply to a tumor or one or morecancer cells, promoting an immune response against one or more cancercells or a tumor, preventing or inhibiting the progression of a cancer,or increasing the lifespan of a subject with a cancer. Anti-canceragents include, for example, chemotherapy agents (chemotherapy),radiotherapy agents (radiotherapy), a surgical procedure (surgery),immune therapy agents (immunotherapy), genetic therapy agents (genetherapy), hormonal therapy, other biological agents (biotherapy) and/oralternative therapies.

In further embodiments antibiotics can be used in combination with thepharmaceutical composition of the present invention to treat and/orprevent an infectious disease. Such antibiotics include, but are notlimited to, amikacin, aminoglycosides (e.g., gentamycin), amoxicillin,amphotericin B, ampicillin, antimonials, atovaquone sodiumstibogluconate, azithromycin, capreomycin, cefotaxime, cefoxitin,ceftriaxone, chloramphenicol, clarithromycin, clindamycin, clofazimine,cycloserine, dapsone, doxycycline, ethambutol, ethionamide, fluconazole,fluoroquinolones, isoniazid, itraconazole, kanamycin, ketoconazole,minocycline, ofloxacin), para-aminosalicylic acid, pentamidine,polymixin definsins, prothionamide, pyrazinamide, pyrimethaminesulfadiazine, quinolones (e.g., ciprofloxacin), rifabutin, rifampin,sparfloxacin, streptomycin, sulfonamides, tetracyclines, thiacetazone,trimethaprim-sulfamethoxazole, viomycin or combinations thereof.

More generally, such an agent would be provided in a combined amountwith the expression vector effective to kill or inhibit proliferation ofa cancer cell and/or microorganism. This process may involve contactingthe cell(s) with an agent(s) and the pharmaceutical composition of thepresent invention at the same time or within a period of time whereinseparate administration of the pharmaceutical composition of the presentinvention and an agent to a cell, tissue or organism produces a desiredtherapeutic benefit. This may be achieved by contacting the cell, tissueor organism with a single composition or pharmacological formulationthat includes both the pharmaceutical composition of the presentinvention and one or more agents, or by contacting the cell with two ormore distinct compositions or formulations, wherein one compositionincludes the pharmaceutical composition of the present invention and theother includes one or more agents.

The terms “contacted” and “exposed,” when applied to a cell, tissue ororganism, are used herein to describe the process by which thepharmaceutical composition and/or another agent, such as for example achemotherapeutic or radiotherapeutic agent, are delivered to a targetcell, tissue or organism or are placed in direct juxtaposition with thetarget cell, tissue or organism. To achieve cell killing or stasis, thepharmaceutical composition and/or additional agent(s) are delivered toone or more cells in a combined amount effective to kill the cell(s) orprevent them from dividing.

The administration of the pharmaceutical composition may precede, beco-current with and/or follow the other agent(s) by intervals rangingfrom minutes to weeks. In embodiments where the pharmaceuticalcomposition of the present invention, and other agent(s) are appliedseparately to a cell, tissue or organism, one would generally ensurethat a significant period of time did not expire between the times ofeach delivery, such that the pharmaceutical composition of the presentinvention and agent(s) would still be able to exert an advantageouslycombined effect on the cell, tissue or organism. For example, in suchinstances, it is contemplated that one may contact the cell, tissue ororganism with two, three, four or more modalities substantiallysimultaneously (i.e., within less than about a minute) as thepharmaceutical composition of the present invention. In other aspects,one or more agents may be administered within of from substantiallysimultaneously, about 1 minute, to about 24 hours to about 7 days toabout 1 to about 8 weeks or more, and any range derivable therein, priorto and/or after administering the expression vector. Yet further,various combination regimens of the pharmaceutical composition of thepresent invention and one or more agents may be employed.

The following examples are provided to further illustrate theembodiments of the present invention, but are not intended to limit thescope of the invention. While they are typical of those that might beused, other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

EXAMPLE 1 Construction of a Replicating HIV-1 Virus Expressing LMP1 orLMP1-CD40

Using recombinant DNA techniques, the coding sequence of LMP1 (1,161 bpencoding 387 amino acids, shown schematically in FIG. 1) is cloned intothe pNL4-3/BaL construct. The resulting plasmid clone is shownschematically in FIG. 2 and is named “NL4-3-BAL-LMP1-IRES” (SEQ ID NO:1). Similarly, LMP1-CD40 (shown schematically in FIG. 1) is cloned intopNL4-3/BaL to create the plasmid shown schematically in FIG. 2 and named“NL4-3-BAL-LMP1-CD40-IRES” (SEQ ID NO: 2). This construct contains theN-terminus of Raji EBV LMP1 (amino acids 1-190) fused in frame with theC-terminus from human CD40 (amino acids 220-277). The sequence of LMP1is provided in SEQ ID NO: 11 and the cytoplasmic fragment or portion ofhuman CD44 is provided in SEQ ID NO: 12.

Following plasmid purification and confirmation by sequencing, theplasmids are used to create live virus by transfecting them into 293Tcells using lipofectamine. 48 hours later, the supernatants arecollected, aliquoted, and stored frozen at −80° C. until use. Infectioustiters are determined using a CD4/LTR-beta-galactosidase HeLa indicatorcell line.

EXAMPLE 2 Macrophages Infected by LMP1- and LMP1-CD40-expressing HIV-1are Stimulated to Make Cytokines

Human blood monocytes are isolated using anti-CD14 immunomagnetic beads(Miltenyi Biotech) and cultured in 48-well plates for 10 days inRPMI1640 supplemented with 10% autologous serum and 2 mM L-glutamine. Atthe end of this period, the non-adherent cells are removed by washingand HIV-1 is added to each well for 4 hours. Following 5 washes withmedia, the cultures are incubated for an additional 8 days, followingwhich the supernatants are assayed for cytokines by enzyme-linkedimmunosorbent assay (ELISA) (R&D Systems) and viral p24 Gag ELISA(Coulter).

As shown in FIG. 3, LMP-expressing HIV-1 strongly stimulates theproduction of MIP-1β and IL-8 by macrophages. LMP-CD40-expressing HIV-1does not stimulate MIP-1 beta production but does stimulate a smallamount of IL-8 production, as shown in FIG. 3. Both viruses replicateless well than unmodified NL4-3/BaL, as judged by the release of p24 Gagantigen.

EXAMPLE 3 Macrophages Infected by LMP1- and LMP1-CD40-expressing VirusRelease Higher Levels of Immunostimulatory Cytokines but Did Not ProduceHigher Levels of Immunosuppressive IL-10

In an experiment similar to Example 2, infected macrophages are culturedfor 9 days after infection before having their supernatants assayed forIL-8, IL-1β, IL-6, IL-12p70, and TNFα. As shown in FIG. 4, theLMP-1-expressing HIV-1 strongly stimulates the production of all ofthese immunostimulatory cytokines. Importantly, however, there is noincrease in the production of the immunosuppressive cytokine, IL-10.Macrophage infection by LMP1-CD40-expressing HIV-1 leads to a similarprofile of cytokine production. These results are gratifying becausethey show for the first time that LMP1 and LMP1-CD40 have effects onmacrophages. Overall, these data indicate that the LMP1 and LMP1-CD40multimerizing-cytoplasmic signaling cassettes promote immunostimulationrather than immunosuppression.

EXAMPLE 4 Dendritic Cells are also Stimulated by Infection with LMP1-and LMP1-CD40-expressing HIV-1

Monocyte-derived dendritic cells (DCs) are prepared from CD14+monocytesby culture in GM-CSF and IL-4 containing media for 6 days. Then the DCsare exposed to virus. As shown in FIG. 5, LMP1-expressing HIV-1stimulates DCs to produce IL-8, IL-1β, TNFα, and IL-6. In contrast,LMP1-CD40-expressing HIV-1 only stimulates DCs to produce IL-8. Theseresults are gratifying because they show for the first time that LMP1and LMP1-CD40 have effects on DCs. Furthermore, these data show thatHIV-1 can be engineered to express these multimerizing-cytoplasmicsignaling cassettes in a functional manner.

EXAMPLE 5 Dendritic Cells Produce a Range of Cytokines FollowingInfection by LMP1- and LMP1-CD40-expressing HIV-1

Monocyte-derived dendritic cells (DCs) are exposed to viruses similarlyto Example 4. As a positive control, a cytokine mix MIMIC™ (Clontech,Palo Alto, Calif.) is used. The MIMIC™ used includes a DC maturationcocktail composed of IL-1β+IL-6+TNF-α+PGE₂. As shown in FIG. 6, assaysof supernatants by ELISA (R&D Systems) 4 days after infection showedthat LMP1-expressing HIV-1 stimulates DCs almost as well as the MIMIC™used with respect to IL-8, IL-1β, IL-6, and TNFα production.Surprisingly, LMP1-expressing HIV-1 stimulated DCs to make IL-12p70bioactive heterodimer, which even the MIMIC™ used is unable tostimulate. Importantly, whereas the MIMIC™ used also stimulates theproduction of IL-10, an immunosuppressive cytokine, IL-10 production isnot increased by LMP1-expressing HIV-1 infection. This indicates thatLMP1-expressing HIV-1 is a better DC stimulus than the MIMIC™ used, thestandard for DC stimulation in vitro. Note that LMP1-CD40-expressingHIV-1 is less active in this regard.

EXAMPLE 6 LMP1-CD40-Expressing HIV-1 Stimulates the Antigen-PresentingAbility of Human DCs

The mixed leukocyte reaction (MLR) is a classic test of alloantigenpresentation. By measuring the expression of ki67, a cell cycle marker,in CD4+ and CD8+ T cells, LMP1-CD40-expressing HIV-1 is found tostimulate DCs to increase both CD4+ and CD830 responses (see FIG. 7).This indicates that LMP1-CD40-expressing HIV-1 is an immunostimulatoryvirus that promotes immune responses by T cells.

EXAMPLE 7 Dendritic Cells can also be Stimulated by a Single-Cycle SIV(scSIV) Expressing LMP1 or LMP1-CD40

In preparation for a trial in rhesus macaques, the LMP1 and LMP1-CD40cassettes are cloned into Simian immunodeficiency virus (SIV). For addedsafety, the SIV used contained mutations in the protease and integrasegenes (see FIG. 8), which prevents the production of progeny virus. Thissingle-cycle SIV (scSIV) is still surprisingly stimulatory for human DCswhen it included a LMP1 or LMP1-CD40 expression cassette (see FIG. 9).This type of single-cycle virus serves as the prototype for a new kindof Human immunodeficiency virus (HIV) vaccine. Similarreplication-defective viruses can be constructed in many viral systemsleading a new series of live, attenuated, but highly immunostimulatoryviral vaccines.

EXAMPLE 8 LMP1-Expressing Single-Cycle SIV is a Self-Adjuvanting Virusfor Anti-SIV Immune Responses

The present invention provides that single-cycle SIV (scSIV) can carryits own antigens into immune responses and a multimerizing-intracellularsignaling cassette can act as an adjuvant for enhancing immuneresponses. Human monocyte-derived dendritic cells (DCs) are studied invitro, where the DCs are exposed to the scSIV viruses for 4 days, andthen co-cultured with human T cells for another 12 days. After theco-culture period, the cells are transferred to the wells of an EnzymeLinked Immuno-Spot (ELISPOT) plate and stimulated with a pool of SIV Gag15-mer peptides. This assay provides a rigorous evaluation ofantigen-presenting function and T cell responses, given that the T cellsare naïve for Gag antigen on day 0. Thus, scSIV-LMP1 induces asignificant increase in Gag-specific interferon gamma responsive T cells(p<0.01 comparing scSIV-LMP1 to scSIV-EGFP). These data and similarresults with HIV-LMP1 demonstrate that scSIV-LMP1 is immunostimulatoryand effectively self-adjuvanting by the inclusion of the LMP1multimerizing-intracytoplasmic signaling cassette. This surprisingresult is the prototype for a range of viruses, vectors, and tumor cellswhere the inclusion of a multimerizing-intracytoplasmic signalingcassette can be used to induce a strong immune response to the antigensthat are co-extensive in space and time—i.e. self-adjuvanting microbialand/or tumor vaccines.

EXAMPLE 9 LMP1 and LMP1-CD40 Can also be Introduced into ModifiedVaccinia Ankara (MVA)

MVA is a widely studied viral vector. As shown in FIG. 11, LMP1 iscloned into the pLW-44 transfer plasmid under the control of the mH5promoter. The P11 promoter drives the expression of GFP. The sequence ofpLW-44 LMP1 is provided in SEQ ID NO: 13 and the sequence of pLW-44LMP1_CD44 is provided in SEQ ID NO: 14.

Chicken embryo fibroblasts (CEF) are infected with wild-type MVA at anMOI of 0.05 (1 well of a 6 well plate). Then, 1 and half hours later,each well is transfected with 2 μg of transfer plasmid using 10 μl ofLipofectamine 2000 (Gibco) in Optimem (Gibco) per manufacturer'sinstructions, and incubated at room temperature for 20 minutes.

The cultures are incubated for three days using two media changes andfinally the infected cells are collected by scraping and freeze-thawedto release viruses. To identify the recombinant viruses, the stock isdiluted and used to infect fresh CEF cells which are covered with amethyl-cellulose overlay, and GFP-containing plaques are identifiedusing a fluorescent microscope.

The recombinant viruses are plaque purified 3 times, and then expandedby growth in CEF bulk cultures. Polymerase chain reaction (PCR) confirmsthat LMP1 and LMP1-CD40 have been successfully introduced into thereplicating viruses. However, MVA containing LMP1-CD40 grows poorly andis unstable, perhaps reflecting an activity of this construct on CEFcells that antagonizes the growth of MVA.

FIG. 28 shows enhanced dendritic cell (DC) maturation by ModifiedVaccinia Ankara (MVA) virus containing LMP1 or LMP1-CD40 adjuvant genecassette.

Overall, this example illustrates that the LMP1 and LMP1-CD40 cassettescan be successfully transferred to a wide range of viruses and microbes,or inserted into viral vectors. This makes these adjuvant-like sequencesvaluable as a means of enhancing the immunogenicity of these agents.

EXAMPLE 10 Construction of LMP1 and LMP-1CD40 Adjuvant Gene Cassettes

LMP1, a constitutively active analog of CD40, is herein provided tofunction as a molecular activator of dendritic cells and macrophagesthat can be used as a powerful vaccine adjuvant.

HIV-1 does not significantly activate cellular immunity, which has madeit difficult to use attenuated forms of HIV-1 as a vaccine. In contrast,Epstein-Barr Virus (EBV) induces robust T cell responses in mostinfected individuals, perhaps because this virus contains LatentMembrane Protein-1 (LMP1), a viral mimic of CD40 which is a keyactivating molecule for dendritic cells (DCs) and macrophages.Consequently, studies have been conducted using LMP1 and LMP1-CD40, arelated construct formed by replacing the intracellular signaling domainof LMP1 with that of CD40. Upon electroporation into DCs, LMP1 andLMP1-CD40 mRNAs are sufficient to upregulate costimulatory molecules andproinflammatory cytokines, indicating that these molecules can functionin isolation as adjuvant-like molecules. As a first step toward animproved HIV vaccine, LMP1 and LMP1-CD40 are introduced into an HIV-1construct to produce virions encoding these proteins. Transduction ofDCs and macrophages with these viruses induced morphological changes andupregulated costimulatory molecules and cytokine production by thesecells. HIV-LMP1 enhances the antigen-presenting function of DCs asmeasured in an in vitro immunization assay. The present inventionprovides that LMP1 and LMP1-CD40 are portable gene cassettes with strongadjuvant properties that can be introduced into viruses like HIV that bythemselves are insufficient to induce protective cellular immunity.

Since Jenner's original description of vaccinia, an attenuated form ofthe smallpox virus, there has been continued interest in using virusesas vaccines. However, whereas viral vaccines like yellow fever 17Dinduce strong protective immunity; viral vaccines for other viruses likeRSV do not. This difference has been explained by the presence orabsence of virus-encoded immunostimulatory: yellow fever 17D stronglyactivates toll-like receptors (TLRs) whereas RSV fails to do so. Theseexamples suggest that viral vaccines are more likely to be effective ifthey carry within them their own adjuvant-like immunostimulatorymolecules.

In this regard, it is notable that HIV-1 is largely deficient in directimmunostimulatory qualities. HIV-1 infection of cultured myeloiddendritic cells (DCs), for example, does not induce DC immunostimulatoryfunctions. Instead, HIV-exposed DCs require exogenous stimuli like CD40ligand (CD40L) to become activated. CD40L is normally expressed byactivated CD4+ T cells and provides DCs with the strong CD40 stimulationneeded to generate effective CD830 T cell responses.

Given the importance of including immune-activating molecules in viralvaccines, it has been demonstrated the existence of strong CD8+ T cellimmunity engendered by infection with Epstein-Barr virus (EBV), a gammaherpesvirus (HIIV4). EBV infection is very common and is oftenmanifested clinically as infectious mononucleosis (AIM), a generallyself-limiting disease. Notably, AIM is characterized by an explosion ofCD8+ T cells that recognize EBV antigens. These anti-EBV CD8+ T cellscontribute to the resolution of EBV viremia and persist as memory cellsat remarkably high levels for years. For example, in healthyEBV-seropositive subjects, up to 5.5% of circulating CD8+ T cells areidentified as EBV-reactive using tetramers for just a single peptideepitope. While it has not been fully determined why EBV is soimmunostimulatory in vivo, the functional similarity of the EBV latentmembrane protein-1 (LMP1) to the CD40 receptor has been demonstrated.Indeed, LMP1 has been viewed as a constitutively activated viral mimicof the CD40 receptor. The present invention provides that LMP1 and arelated protein, LMP1-CD40, can be used as molecular adjuvants for DCsand macrophages. The present invention further provides that both LMP1and LMP1-CD40 strongly activated these antigen-presenting cells (APCs).This function is maintained when these molecules are inserted into theHIV-1 genome and the resulting viruses are strongly activating for APCsin vitro. Consequently, the present invention provides that LMP1 andLMP1-CD40 can serve as portable gene cassettes with adjuvant-likequalities that can be used to improve the cellular immune response toviral vaccines.

Cells and reagents: Venous blood is obtained from Continental BloodServices, Inc. (Miami, Fla.) as anonymous Buffy Coat donations fromHIV-seronegative donors. Peripheral blood mononuclear cells (PBMCs) areisolated by centrifugation over Ficoll-hypaque and subsequently culturedin RPMI 1640 (Thermo Scientific HyClone, Logan, Utah) supplemented with5% human AB serum (Lonza, Allendale, N.J.) and 10 mM HEPES (Invitrogen,Carlsbad, Calif.) (complete RPMI 1640). 293T HEK cells are cultured inDulbecco's modified Eagle medium (DMEM) supplemented with 10% fetalbovine serum (FBS), 2 mM L-glutamine, and antibiotics (100 U/mlpenicillin and 100 μg/ml streptomycin). TZM-bl cells are obtainedthrough the National Institutes of Health AIDS Research and ReferenceReagent Program (catalogue no. 8129, contributed by John C. Kappes andXiaoyun Wu), cultured in RPMI 1640 medium (supplemented with 10% FBS, 2mM L-glutamine, and antibiotics), and used to quantify infectious HIVvirions.

Plasmid DNAs: Plasmid DNA encoding enhanced green fluorescent protein(EGFP) is obtained from Dr. Chelsa Spina. pNL-BaL (termed pHIV in thefigure legends) is a macrophage-tropic proviral clone of pNL4-3containing the CCR5-utilizing envelope from HIV-1BaL. Plasmids are grownin E. coil DH 5α and isolated using a Plasmid Maxi Kit (QIAGEN,Valencia, Calif.). To reduce the presence of exogenous stimulatingmaterials, the plasmids are further cleaned using Triton X-114extraction.

Construction of LMP1 and LMP1-CD40 adjuvant gene cassettes: cDNA isprepared from Raji B cells (American Type Culture Collection, Manassas,Va.) and the LMP1 sequence of EBV is isolated by polymerase chainreaction (PCR) and found to be identical to the reference sequence(GenBank M58153.1). Related to LMP1 is an artificial fusion proteincontaining the multimerizing, membrane-associated N-terminus of LMP1conjoined with the intracytoplasmic domain of CD40, i.e., LMP1-CD40.This membrane-associated intracellular fusion protein mimics theconstitutive signaling of CD40 without the need for an external ligand.LMP1-CD40 is constructed by fusing DNA encoding the N-terminal 190 aminoacids of LMP1 (beginning with the ATG start codon) with the C-terminal58 amino acids of CD40 followed by a TGA translational stop codon. Forthe preparation of LMP1 and LMP1-CD40 mRNA described below, thesesequences are cloned into the pGEM-4Z/A64 vector.

Preparation of HIV-1 virions expressing LMP1 or LMP1-CD40: Levy et al.(2004) Proc Natl Acad Sci USA 101: 4204-9, has described the design ofreplication competent HIV-1 proviral clones in which exogenous codingsequences such as EGFP can be inserted just 5-prime to nef, followed byan internal ribosome entry site (IRES) that allows continued translationof the natural Nef protein (FIG. 2). Starting with the macrophage-tropicpNL-BaL proviral clone, overlap PCR was used to construct plasmidsexpressing either EGFP (pHIV-EGFP), LMP1 (pHIV-LMP1), or LMP1-CD40(pHIV-LMP1-CD40). The present invention provides that these exogenousgenes are expressed in nef-spliced mRNAs where the nef-spliced mRNAs arevery strongly expressed and are the predominant mRNA species inmacrophages during the first 24 hours following HIV-1 infection. Thisdesign maximizes the expression of these exogenous genes, whereas otherprovirus designs would be expected to result in more modest expressionof these transgenes.

EXAMPLE 11 Preparation of DCs and Macrophages and Virus Transduction

To isolate monocytes from PBMCs, cells are first adhered to T175 plasticflasks (Coming-Costar, Cambridge, Mass.) for 24 hours in RPMI 1640containing 10% heat-inactivated human AB serum. Following washing toremove non-adherent cells, the adherent monocytes are harvested bygentle scraping and transferred into 24-well at a density of 1×10⁶cells/well. To allow differentiation into monocyte-derived macrophages(macrophages), the cultures are incubated for an additional 7 days. Toprepare monocyte-derived dendritic cells (DCs), the monocytes arecultured in 800 U/ml GM-CSF and 500 U/ml IL-4 (R & D Systems, Inc.,Minneapolis, Minn.) for 5 days, adding fresh Granulocyte macrophagecolony-stimulating factor (GM-CSF) and IL-4 on day 3.

Lentiviral transduction of DCs and macrophages and flow cytometry:Infections are initiated by aspirating the supernatant media from thewells containing 10⁶ DCs or macrophages and then adding 100 μl/well ofmedia containing 100 ng p24 equivalents of HIV (calculated to be amultiplicity of infection of 0.1) followed by culture at 37° C. for 4hours. Next the cells are washed twice with RPMI medium and then fedwith 2 ml of complete RPMI 1640 followed by incubation at 37° C. for upto 8 days. The culture supernatants of transduced DCs or macrophages arecollected at different time points and stored at −80° C. For flowcytometry, macrophages are stained on the plates and then harvested byscraping, while DCs are first harvested by gentle scraping and thenre-suspended for staining. Cells are washed in fluorescence-activatedcell sorter buffer (PBS supplemented with 3% fetal calf serum and 0.02%sodium azide) and then stained by fluorochrome-conjugated antibodies.Flow cytometry is performed using a LSRII bioanalyzer (Becton Dickinson)and analyzed with the FlowJo software program (Tree Star, San Carlos,Calif.).

Measurement of DC and macrophage activation by cytokine assays andRT-PCR for chemokine mRNA: For cytokine measurements, supernatants arecollected from DC and macrophage cultures either 48 hours after mRNAelectroporation or 7 days after virus infection and stored at −80° C.until assay. Concentrations of IL-8, IL-6, IL-1β, TNFα, IL-10 andIL-12p70 are measured by cytometric bead array (CBA) (BD Biosciences,San Jose, Calif.) according to manufacturer's instructions.Additionally, macrophages are infected for 7 days and then analyzed byRT-PCR to measure steady-state levels of CCL3 (MIP-1α), CCL4 (MIP-1β)and CCL5 (RANTES).

In vitro immunization assay for T cell responses: DCs from an HIVseronegative donor are exposed to different HIV viruses for 6 days whichintroduced HIV antigens into these antigen-presenting cells. The DCs arethen incubated with autologous T cells for 12-days in the presence of 5μM nevirapine to prevent HIV infection of any added CD4+ T cells. IL-2(5 U/ml) is added on day 3 and day 8. Following DC-T cell coculture,antigen-specific T cell responses are quantified by IFN-γ ELIPOT assay.Cultured cells (10⁵/well) are added to 96-well multiscreen plates(Millipore, Bedford, Mass.) that have been precoated with 0.5 g/ml ofanti-IFN-γ monoclonal antibody (BD Biosciences, San Jose, Calif.). Apool of 15-mer Gag HIV-1 Clade B consensus peptides (AIDS ReagentProgram Cat. # 8117) is added at a final concentration of 5 μg/ml. As anegative control, cells are also cultured without peptide. Plates areincubated overnight at 37° C., 5% CO₂ and developed. The numbers ofspots are determined using an automated ELISPOT plate reader (CTLTechnologies, Cleveland, Ohio), and the spot-forming counts (SFC) arecalculated by subtracting the negative-control wells (mean plus 3standard deviations). A value of 55 SFC/10⁶ PBMC or greater aftersubtraction of background is considered positive.

Statistics: Data are analyzed using PRISM 4.0 (GraphPad Software, LaJolla, Calif.) and expressed as the mean ± SEM. Statistical comparisonsare analyzed by Student's t test. A P value of 0.05 is consideredstatistically significant.

Electroporation of DCs with LMP1 and LMP1-CD40 mRNAs: Plasmidscontaining the coding sequences for LMP1 (pLMP1) and LMP1-CD40(pLMP1-CD40) are linearized by digestion with NdeI downstream of thepoly A tail and then in vitro transcribed with T7 polymerase tosynthesize capped mRNA with additional poly (A) tail using the mMessagemMachine kit (Ambion, Austin, Tex.). DC cultures are re-suspended at aconcentration of 10⁷ cells/ml in Opti-MEM medium (Invitrogen), followingwhich 0.2 ml is added to a 4 mm electroporation cuvette along with 10 μgof mRNA. Transfection is performed by electroporation using a GenePulser (BioRad Laboratories, Hercules, Calif.) set to 350 V, 150 μF.Following electroporation, the cells are plated in 6 well plates incomplete RPMI 1640 and cultured for another 48 hours.

Preparation and characterization of virus stocks: To prepare virusesfrom the proviral clones, 5Δ10⁶ 293T cells are first plated in 100 mmdiameter dishes in complete DMEM. The next day, 5 μg of either pHIV,pHIV-EGFP, pHIV-LMP1, or pHIV-LMP1-CD40 plasmid DNA is transfected usingthe GenJet Plus Transfection Reagent according to the manufacturer'sinstructions (Signagen Laboratories, Iamsville, Md.). The cell culturemedia is replaced 24 hours later, and virus-containing supernatants arecollected after a further 24 hours in culture. Cell debris is removed bycentrifugation and the supernatants are filtered through a 0.45 μmembrane (Millipore, Bedford, Mass.). Virus stocks are titered using theTZM-bl cell assay and expressed as the 50% tissue culture infectiousdose (TCID₅₀) as calculated using the Reed and Muench formula. For theexperiment shown in FIG. 14, the virus stock is further purified usingimmunomagnetic beads (μMACS Virus Isolation Kit, Miltenyi Biotec,Auburn, Calif.), which eliminates the possible confounding effects ofnon-virion soluble molecules or cellular debris.

Western blotting for LMP1 and LMP1-CD40 expression is used to confirmthat HIV-LMP1 and HIV-LMP-CD40 viruses lead to expression of theirrespective proteins in 293 T cell viral lysates. SDS lysis buffer isadded to cells and run on a 10% SDS-PAGE gel, following by transfer tonitrocellulose membranes, blocking with 5% evaporated milk with 0.2%Tween-20 in PBS. Membranes are stained overnight at 4° C. with eithermouse anti-EBV LMP1 monoclonal antibody (3H2104,a,b,c, 1:100 dilution,Santa Cruz Biotechnology, Santa Cruz, Calif.) or rabbit anti-human CD40polyclonal antibody (C-20, 1:200 dilution, Santa Cruz Biotechnology,Santa Cruz, Calif.). Membranes are then washed with PBS-0.2% Tween-20and incubated with either horseradish peroxidase (HRP)-conjugated goatanti-mouse or anti-rabbit antibody (Pierce, Rockford, Ill.) at a 1:5,000dilution in blocking buffer. Then the membranes are washed and incubatedin HRP substrate (Pico chemiluminescence; Pierce), placed on Whatman 3MMfilter paper and exposed to film (BioMax; Kodak, Rochester, N.Y.). Bandsof the predicted sizes are observed for p24 Gag (24 kDa), LMP1 (39 kDa),LMP1-CD40 (26 kDa).

Measurement of DC and macrophage activation by flow cytometry: To assessthe maturation and activation of DCs and macrophages, the conjugatedmonoclonal antibodies against the following surface molecules are used:CD40 (clone 5C3, BD Pharmingen, San Diego, Calif.); CD80 (clone L307.4,BD Pharmingen, San Diego, Calif.); CD83 (clone HB15, BD Pharmingen, SanDiego, Calif.); and CD86 (clone 2331, BD Pharmingen, San Diego, Calif.);HLA-DR (clone L243, BD Pharmingen, San Diego, Calif.); CCR7 (clone 3D12,BD Pharmingen, San Diego, Calif.).

Quantitative RT-PCR for chemokine mRNAs: Macrophages are infected for 7days and then analyzed by RT-PCR to measure steady-state levels of CCL3(MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES). Total RNA is prepared usingRNeasy kit (Qiagen Inc., Valencia, Calif.), treated with RNAse-freeDNAse (Roche Molecular Biochemicals, Indianapolis, Ind.) and used astemplate in an RT-PCR assay. RNA is reverse transcribed in a 20 μlreaction containing 0.1 μg of total RNA, 0.1 μg of oligo(dT), 200 U ofreverse transcriptase (Finnzymes, Finland) and 0.2 μM each of dATP,dCTP, dGTP and dTTP. After incubation at 40° C. for 1 hour to generatecDNA, aliquots are analyzed by real-time PCR using the Power SYBR GreenSupermix (Applied Biosystems). The following primers are used: CCL3(MIP-1α)-specific primers, 5′-GTCTGTGCTGATCCCAGTGA-3′ (forward) (SEQ IDNO: 5) and 5′-TTGTCACCAGACGCGGTGTG-3′ (reverse) (SEQ ID NO: 6); CCL4(MIP-1β)-specific primers, 5′-GTCTGTGCTGATCCCAGTGA-3′ (forward) (SEQ IDNO: 7) and 5′-GGACACTTATCCTTTGGCTA-3′ (reverse) (SEQ ID NO: 8); CCL5(RANTES)-specific primers, 5′-CCGCGGCAGCCCTCGCTGTCATCC-3′ (forward) (SEQID NO: 9) and 5′-CATCTCCAAAGAGTTGATGTACTCC-3′ (reverse) (SEQ ID NO: 10).For noiuialization, GAPDH and β-actin real-time PCR is carried out onthe same samples. Normalized mRNA levels for each transcript arecalculated as (1/2ΔCt×1,000), where ΔCt value=Ct (test mRNA)-Ct (GAPDHmRNA). To control for contamination with genomic DNA, parallelamplifications are performed in the absence of reverse transcriptase andare uniformly negative.

EXAMPLE 12 EBV LMP1 Activates Dendritic Cells and Functions as aMolecular Adjuvant When Incorporated into an HIV Vaccine

Preparation of LMP1 and LMP1-CD40: The Raji B cell line is used for thepreparation of LMP1 cDNA which encoded a 387 amino acids protein. Toprepare LMP1-CD40, PCR gene construction techniques are used to fuse thenucleotides for the N-terminal 190 AA of LMP1 (GenBank M58153.1) withthe C-terminal cytoplasmic tail of human CD40 (residues 220-277, GenBankNM_001250). The resulting proteins are depicted in FIG. 1. Notably, LMP1N-terminal residues form a domain with six transmembrane regions thatself-associates in the plane of the membrane, thereby clustering thecytoplasmic tails of these proteins. On the intracellular side of theplasma membrane, the clustered signaling domains recruit adaptermolecules such as TRAFs to initiate downstream signaling events. CD40Lis not needed to induce clustering of these receptor mimics and as aresult both LMP1 and LMP1-CD40 are constitutively active.

Electroporation of mRNAs for LMP1 or LMP1-CD40 activate DCs: Both LMP1and LMP1-CD40 are known to be active in B cells and certain epithelialcells, but their effects on DCs are not previously known. Consequently,in vitro transcribed mRNA for LMP1 or LMP1-CD40 is electroporated intoDCs. For comparison, control cultures included DCs electroporatedwithout added mRNA (mock) and DCs electroporated with mRNA for enhancedgreen fluorescent protein (GFP), an inactive protein. As shown in FIG.12A, LMP1 or LMP1-CD40 mRNA alone is sufficient to upregulate CD40,CD80, and the CD83 maturation marker on DCs as measured by flowcytometry. CD86 expression, however, is not significantly changed,reflecting its independent regulation in these cells. In parallel withthese membrane changes, DCs are also stimulated to secrete cytokines into the media as measured by cytometric bead assay (CBA) (FIG. 12B). BothLMP1 and LMP1-CD40 induce sizable amounts of TNFα, IL-6, and IL-8. Atrace amount of IL-1β is produced as well, but essentially no IL-10 orIL-12p70 is made. The lack of IL-12p70 production is consistent withreports that CD40 stimulation alone is insufficient to cause IL-12p70production in the absence of a second stimulus such as bacterialendotoxin/LPS or other TLR agonist. Thus, the data demonstrate that LMP1and LMP1-CD40 are sufficient to activate many important DC functionseven when used alone as unvectored isolated gene cassettes.

Introduction of LMP1 or LMP-1-CD40 into an HIV-1 proviral construct:Given the DC-activating effects of LMP1 and LMP1-CD40 alone, the presentinvention provides that they can retain this function when produced inthe context of a viral vector, e.g., HIV-1. Consequently, a proviralclone of pNL4-3 is obtained that has been modified to contain themacrophage-trophic envelope gene of HIV-1BAL yielding a CCR5-utilizingvirus that can infect DCs and macrophages. This virus is furtherengineered using the design of Levy et al. (2004) Proc Natl Acad Sci USA101: 4204-9, where exogenous coding sequences can be inserted into theHIV-1 genome just 5-prime to nef, followed by an internal ribosome entrysite (IRES) that allows continued translation of the Nef protein (FIG.2). This results in infection-competent, replicating viruses thatexpress either LMP1 (HIV-LMP1) or an LMP1-CD40 fusion (HIV-LMP1-CD40).When the plasmids encoding HIV-LMP1 or HIV-LMP1-CD40 are transfectedinto 293 cells, the synthesis of correctly sized LMP1 and LMP1-CD40proteins is demonstrated by Western blotting (FIG. 16).

HIV-LMP1 and HIV-LMP1-CD40 stimulate DCs and macrophages to upregulateimmunologically important cell surface molecules. DCs and macrophagesare transduced with HIV-LMP1 or HIV-LMP1-CD40 and cultured for 4 days toallow these viruses to enter the cells and express virally encodedproteins. At this time point, DCs develop extensive dendritic processessimilar to the changes induced by stimulation with cells expressingmembrane CD40L, Using flow cytometry analysis, both engineered virusesaffect DCs (FIG. 13A) by upregulating CD40, CD80, and CD83, and had onlyminor effects on the expression of CD86. A similar pattern of DCactivation is shown when LMP1 or LMP1-CD40 mRNAs are electroporated intoDCs (FIG. 12A), indicating that the specific functions of thesemolecules are unchanged by incorporation into a viral vector. Inaddition, HLA-DR (an MHC-II molecule) and CCR7 are also studied. WhileHIV-LMP1 and HIV-LMP1-CD40 do not affect HLA-DR expression by DCs andmacrophages, both of these engineered viruses upregulate CCR7expression. Since CCR7 controls the entry of cells into lymph nodes, thepresent invention provides that the CCR7 upregulation produced byHIV-LMP1 or HIV-LMP1-CD40 can allow these transduced cells to migrate tolymph nodes where they can in turn present the viral antigens encoded bythe HIV vector.

Similarly, macrophages are strongly stimulated by HIV-LMP1 andupregulated CD40, CD80, CD83, and CD86 expression (FIG. 13B). Incontrast, HIV-LMP1-CD40 is not a strong stimulator of macrophages,although CD86 is modestly upregulated. These changes are mirrored in themorphological changes observed in these cultures. HIV-LMP1 exposedmacrophages undergo extensive cell-cell clumping as previously reportedusing CD40L stimulation. In contrast, HIV-LMP1-CD40 induces a moremodest degree of clumping. Thus, for these cell surface proteins, itappears that LMP1 is strongly active in both DCs and macrophages whereasLMP1-CD40 is more active in DCs than macrophages.

HIV-LMP1 and HIV-LMP1-CD40 stimulate DCs and macrophages to producecytokines and chemokines. As further studies of the DCs and macrophagescultures shown in FIG. 13, culture supernatants from day 4 are studiedfor the secretion of cytokines and chemokines. For DCs (FIG. 14A),HIV-LMP1 significantly increases the production of IL-1β, IL-6, IL-8,IL-12p70, and TNFα. In contrast, HIV-LMP1-CD40 is less active thanHIV-LMP1 for cytokine induction in DCs. As shown in FIG. 14B, HIV-LMP1and HIV-LMP1-CD40 induces macrophages to make IL-6, IL-8, and smallamounts of IL-12p70, IL-1β and TNFα. In contrast, there are nosignificant effects on IL-10 production. Serial measurements made ondays 4, 7, and 10 show that high levels of IL-1β, IL-6, and IL-8 persistin these cultures, whereas IL-10, IL-12p70, and TNFα fall to backgroundlevels by day 10. In addition, steady-state levels of three chemokines(CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES)) are increased inmacrophages infected by HIV-LMP1 and especially HIV-LMP1-CD40,suggesting fine differences in the cell signaling produced by the twoviral constructs (FIG. 17).

HIV-LMP1 and HIV-LMP1-CD40 upregulate the antigen-presenting functionsof DCs in an in vitro immunization assay. To provide an initialindicator of the effects of HIV-LMP1 and HIV-LMP1-CD40 on immuneresponses to the HIV viral vector, an in vitro immunization model isused. This assay relies on the fact that T cells from HIV-uninfectedindividuals have a low but definite frequency of reactivity to HIVantigens. Consequently, when purified autologous T cells are cultured onDCs exposed to these viruses and supplemented with IL-2, the rareanti-HIV T cells in the population can be expected to respond to the HIVantigens in the DCs. Using an IFN-γ ELISPOT assay and a pool of HIV Gagpeptides, exposure to HIV-LMP1 is found to significantly enhance the invitro response to Gag antigen (FIG. 15). In contrast, HIV-LMP1-CD40 isno better than control HIV-GFP in this assay. Thus, in some embodiments,HIV-LMP1 is a strong immunogen in vivo and it may be more active thanHIV-LMP1-CD40.

EXAMPLE 13 LMP1 and LMP1-CD40 can be Used as Immune Stimulators for DCsand Macrophages

The starting point is the clinical observation that EBV infectionelicits extremely strong CD8+ T cell responses directed against its ownantigens and immunological studies showing that this virus contains aviral mimic of the CD40 receptor, LMP1, which is constitutively active.LMP1 is known to be active in B cells, but its effects on DCs andmacrophages had not been previously reported. The present inventionprovides that LMP1 and LMP1-CD40 are activators of these cells and canbe inserted into viral vectors, for example HIV, to convert a weaklyimmunostimulatory virus into a strong immune stimulator that can be usedas the basis for a candidate vaccine.

The key finding is that LMP1 and LMP1-CD40 can be used in isolation tostimulate DCs and macrophages. This can be accomplished byelectroporating mRNAs for these genes into these cells in vitro andobserving the pattern of activation that resulted. Costimulatoryproteins on the cell surface are upregulated and cytokine production isinduced. These data confirm prior reports that the transfection ofplasmid DNA encoding LMP1 has strong CD40L-like activating effects on Bcells and B cell-derived tumor lines. Extending these prior findings,the present example shows that LMP1 and LMP1-CD40, separate from anyvector containing them, can act alone as completely functionalimmunostimulatory molecules capable of activating DCs and macrophages.

As adjuvant molecules, LMP1 and LMP1-CD40 offer several advantages:First, LMP1 is one of the few immunostimulatory proteins that do notelicit strong CD8+ T cell responses against itself. While very strongCD8+ T cell responses are directed against the lytic proteins expressedby EBV, CD8+ T cell reactivity against LMP1 is difficult to detect.Thus, DCs and macrophages activated by LMP1 (and presumably alsoLMP1-CD40) are not likely to be rapidly killed by pre-existing orinduced CD8+ T cells recognizing peptides from this protein.

Second, LMP1 and LMP1-CD40 have potential advantages over using CD40L asa molecular adjuvant. Prior studies have shown that the inclusion ofCD40L into Vaccinia, canarypox, adenovirus, lentiviral vectors, and SIVsignificantly improved the immunogenicity of these viruses and viralvectors. In all of these cases, the production of virus or virus-likeparticles (VLPs) from CD40L-expressing cells leads to virions and VLPsbearing functional CD40L on their membrane surface, which can beproblematic. Virions and VLPs bearing surface CD40L can bindindiscriminately to the many cell types that express the CD40 receptor,including B cells and endothelial cells. This binding can havefunctional consequences and CD40L-bearing viruses and VLPs have beenshown to activate bystander B cells and macrophages. This results in apotential for inducing autoimmunity and other deleterious effects. Incontrast, LMP1 and LMP1-CD40 are cell-associated proteins thatinternally activate the cells that express them; they do not confer aligand-like ability to activate other cells that come in contact withthe cells that express them. Thus, LMP1 and LMP1-CD40L should notdisturb the cell-targeting qualities built into a viral vector norshould they lead to wide-spread and possibly toxic bystander cellactivation. As a result, the present invention provides that a viralvector bears these molecules to be “self-adjuvanting,” where the tightlinkage in time and space of the vector-expressed antigen to theseadjuvant molecules allows these two moieties to serve as a completeimmunogen, focusing the immune response on the selected antigen.

In addition to LMP1 and LMP-CD40, a chemically controlled method ofmultimerizing the CD40 intracellular signaling domain has beendeveloped. In their system, chemically induced dimerization (CID), abivalent chemical is used to cluster specially modified CD40 domainstethered to the cytoplasmic side of the plasma membrane. Like LMP1 andLMP1-CD40, this approach requires the introduction of gene constructsinto cells either by plasmid transfection or viral transduction. UnlikeLMP1 and LMP1-CD40, the system requires the separate administration ofthe CID crosslinking molecule so that it attains pharmacologicallyadequate tissue levels in vivo. This degree of regulatory control inthis system is attractive, but it can be achieved in other ways. Forexample, the LMP1 and LMP1-CD40 system described herein can be regulatedusing an inducible promoter system. The Tet-On promoter system can beoptimized to create an HIV construct that replicates only in thepresence of doxycycline. Such a conditionally replicating form ofHIV-LMP1 or HIV-LMP1-CD40 can be a safer way of using these vaccinecandidates. Even safer would be to use a form of HIV or lentivirus thatis limited to a single cycle of replication in vivo. The presentinvention provides that any viral vector well known in the art can bemodified to include LMP1 or LMP1-CD40 as molecular adjuvants.

The studies reported herein suggest yet other modifications for improvedvectored vaccines. CD40 stimulation is highly synergistic with Toll-likereceptor (TLR) stimulation for the induction of CD8+ T cell responses,which raises the possibility of combining LMP1 or LMP1-CD40 withconstitutively active forms of TLRs. Also, in vivo studies are needed todetermine if the fine differences between LMP1 and LMP1-CD40 signalingwill result in meaningful differences in vaccine responses.

In conclusion, this example describes two portable genetic adjuvants,LMP1 and LMP1-CD40, which can be used to activate DCs and macrophages.These molecules have considerable potential for strengthening existingvaccines based on attenuated viruses or viral vectors. In particular,they can be especially important for the design of HIV vaccines givenour finding that these genetic adjuvants can be easily introduced intothe genome of this otherwise poorly immunogenic virus.

EXAMPLE 14 Latent Membrane Protein 1 as a Molecular Adjuvant forSingle-Cycle Lentiviral Vaccines

Molecular adjuvants are a promising method to enhance virus-specificimmune responses and protect against HIV-1 infection. Immune activationby ligands for receptors such as CD40 can induce dendritic cellactivation and maturation. The present example provides theincorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or anLMP1-CD40 chimera, into a strain of SIV that is engineered to be limitedto a single cycle of infection.

Full length LMP1 or the chimeric protein LMP1-CD40 is cloned into thenef-locus of single-cycle SIV. Human and Macaque monocyte derivedmacrophages and DC are infected with these viruses. Infected cells areanalyzed for activation surface markers by flow cytometry. Cells arealso analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6,IL-8, IL-12p70 and TNFα by cytometric bead array.

Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produces a broadand potent Th1-biased immune response in human as well as rhesus macaquemacrophages and DC when compared with control virus, Single-cycleSIV-LMP1 also enhances antigen presentation by lentiviral vectorvaccines; illustrating that LMP1-mediated immune activation can enhancelentiviral vector vaccines against HIV-1.

To develop an effective lentiviral vector vaccine against HIV-1infection it may be necessary to focus on enhancing the activation ofdendritic cells, and other professional antigen presenting cells, inorder to maximize the stimulation of virus-specific immune responses.One of the critical events in the induction of immune response is thematuration of DCs and macrophages. Maturing DCs and macrophages undergoa rapid burst of cytokine synthesis and expression of costimulatorymolecules. Dendritic cells then migrate to the T-cell areas of drainingsecondary lymphoid organs to prime naïve T cells and initiate anadaptive immune response. IL-12p70 is secreted by activated macrophagesand DC and stimulates IFN-γ secretion by T lymphocytes and NK cells. Toimprove the efficacy of vaccines, single-cycle SIV vaccines can bedeveloped by incorporating inducers of antigen presenting cellmaturation and cytokine secretion, specifically looking at CD40stimulation and the role of the viral protein LMP1.

LMP1 is an integral membrane protein of Epstein Barr Virus (EBV) with amolecular weight of approximately 63 kDa consisting of three domains.LMP1 expression induces many of the changes associated with EBVinfection and activation of primary B cells, including cell clumping;increased cell surface expression of CD23, CD39, CD40, CD44; decreasedexpression of CD10; and increased expression of the cell adhesionmolecules CD11a (LFA1), CD54 (ICAM1), and CD58 (LFA3). At least foursignaling pathways, namely nuclear factor κB (NF-κB), c-Jun N-terminalkinase (JNK)-AP-1, p38/MAPK (mitogen activated protein kinase), andJanus kinase (JAK)-STAT (signal transducers and activators oftranscription), are implicated in the function of LMP1. Within theC-terminus of LMP1 there are at least two activating regions referred toas CTAR1 and CTAR2 (C-terminal activating region). CTAR1 is locatedproximal to the membrane (amino acids 186-231) and is essential for EBVmediated transformation of primary B cells. CTAR2 (amino acids 351-386)is located at the extreme C-terminus of LMP1 and is required for longterm growth of EBV positive primary B cells. Both CTAR1 and CTAR2 canactivate NF-κB independently. Aggregation of LMP1 within the plasmamembrane is a crucial prerequisite for signaling. LMP1 aggregationappears to be an intrinsic property of the transmembrane domain. Thissignaling is similar to signaling by the tumor necrosis factor receptor(TNFR) CD40. The main difference between LMP1 and the TNFR family isthat LMP1 functions as a constitutively activated receptor and,therefore, does not rely on the binding of an extracellular ligand forcostimulation. Experiments have also evaluated the chimeric moleculeLMP1-CD40, consisting of the LMP1 transmembrane domain and the CD40cytoplasmic tail. These experiments suggest that the LMP1-CD40 chimerais also constitutively active in vitro.

In the present example, LMP1 and LMP1-CD40 genes are incorporated intothe genome of pseudotyped single-cycle SIV viral particles. These genesare expected to enhance the immunogenicity of the virus, therebystimulating antigen presentation by infected APC. The immunogenicity ofSIV-LMP1 and SIV-LMP1-CD40 are evaluated in vitro using human as well asmacaque monocyte-derived DCs and macrophages. The present inventionprovides that LMP1 and LMP1-CD40 significantly enhance the ability ofSIV to activate DCs and macrophages. SIV-LMP1 also enhances the primingof naive Gag-specific T cells in vitro. These results are encouragingfor the clinical evaluation of LMP1 and LMP1 chimeric constructs as anovel class of adjuvant for HIV vaccines and other immunotherapystrategies.

Cells and media: Embryonic kidney (293T) cells are grown at 37° C. under5% CO₂ in Dulbecco's modified Eagle medium (DMEM) supplemented with 10%fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics (100 U/mlpenicillin and 100 μg/ml streptomycin), (referred to as completemedium). Human as well as rhesus macaque peripheral blood mononuclearcells (PBMCs) are prepared by Ficoll-Hypaque density centrifugation andmaintained in RPMI medium (Hyclone, Logan, Utah) supplemented with 5%human serum (Lonza, Allendale, N.J.) and 10 mM HEPES (Invitrogen,Carlsbad, Calif.).

Plasmid Construction: The construct SIVmac239 FS-ΔPR-ΔINEGFP containsmutations in the gag-pol frameshift site (FS) and deletion in theprotease (ΔPR) integrase (ΔIN) coding regions of the pol gene. The Nefcoding region is replaced with GFP. All constructs with theimmunostimulatory genes LMP1 or LMP1-CD40 are cloned by overlap PCR andinserted into the SIVmac239 FS-ΔPR-ΔINEGFP vector using unique Xbal andSacII sites flanking the GFP gene. All viral clones are confirmed by DNAsequencing both before and after ligation into the viral vector. All DNAplasmids are purified with the Qiagen Endo-Free kit and checked forendotoxin levels prior to transfection.

Preparation of viral stocks: Single-cycle virus stocks are prepared byharvesting the supernatant of 293T cells transfected with differentviral plasmids. VSV-G trans-complemented single-cycle SIV is produced byco-transfection of 293T cells with the Gag-Pol expression constructpGPfusion, 5 μg of an expression construct for the Indiana or the NewJersey serotype of VSV-G and a full-length proviral DNA construct foreach scSIV strain. 293T cells are seeded at 5×10⁶ cell per 100-mm dishin cell culture medium (Dulbecco's modified Eagle's medium [DMEM]supplemented with 10% fetal bovine serum [FBS], L-glutamine, penicillinand streptomycin) and transfected the following day with 5 μg of eachplasmid using Genjet plus transfection Reagent (Signagen Laboratories,Iamsville, Md.). Twenty-four hours after transfection, the plates arerinsed twice with serum-free medium and the cell culture medium isreplaced with DMEM supplemented with 10% FBS. Twenty-four hours later,the cell culture supernatant is collected, clarified by centrifugationat 500×g for 10 min, and filtered through a 0.45 μm-pore-size membrane(Millipore, Bedford, Mass.). To prepare high-titre stocks, viralparticles are concentrated by repeated low speed centrifugation usingYM-50 ultrafiltration units (Millipore, Bedford, Mass.). Aliquots (1 mL)of scSIV are cryopreserved at −80° C. and the concentration of virus isdetermined by p27 antigen capture ELISA (Advanced BioScienceLaboratories, Kensington, Md.).

Single-cycle SIV infectivity assays: One million CEM×174 cells areincubated with 100 ng p27 equivalents of scSIV in 100 μl volume for 2hours at 37° C. Cultures are then expanded to a volume of 2 ml in R10medium (RPMI supplemented with 10% FBS, L-glutamine, penicillin andstreptomycin) and incubated in 24-well plates at 37° C. for 4 days.Cells are treated with Fix and Penn reagents (BD Biosciences, San Jose,Calif.) and stained with FITC-conjugated SIV Gag-specific monoclonalantibody (Immunodiagnostics Inc. Woburn, Mass.). After staining, cellsare fixed in 2% paraformaldehyde PBS and analyzed by flow cytometry todetermine the frequency of SIV Gag-positive infected cells.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Westernblotting: Viral particle stocks are run on a 10% sodium dodecylsulfate-polyacrylamide gel (Bio-Rad, Hercules, Calif.). Proteins arethen transferred to nitrocellulose membranes (0.22 μm; GE Osmonics,Minnetonka, Minn.) and blocked (5% milk in PBS-0.2% Tween 20). Themembranes are incubated individually with primary antibody overnight at4° C. These antibodies include the following: (i) 1:100 dilution ofmouse anti-EBV LMP1 monoclonal antibody (3H2104,a,b,c Santa CruzBiotechnology, Santa Cruz, Calif.), (ii) 1:500 dilution of mouseanti-CD40 polyclonal antibody (C-20, Santa Cruz Biotechnology, SantaCruz, Calif.), and (iii) 1:2,000 dilution of mouse anti-Gag p27antibody, obtained through the National Institutes of Health AIDSResearch and Reference Reagent Program (Germantown, Md.) (SIVmac251 Gagmonoclonal [KK64], catalogue no. 2321, from Karen Kent and CarolinePowell). Membranes are washed with PBS-0.2% Tween 20 and incubated withhorseradish peroxidase (HRP)-conjugated goat anti-mouse antibody(Pierce, Rockford, Ill.) at a 1:5,000 dilution in blocking buffer.Following incubation in the secondary antibody, the membranes are washedand then incubated in HRP substrate (Pico chemiluminescence; Pierce).Membranes are placed on Whatman 3MM filter paper and exposed to film(BioMax; Kodak, Rochester, N.Y.).

Preparation and transduction of monocyte-derived macrophages anddendritic cells: PBMC from healthy blood donors (Continental BloodServices, Miami, Fla.) are isolated from buffy coats by densitycentrifugation using Ficoll-Hypaque (Amersham Pharmacia Biotech Inc.,Piscataway, N.J.). Cells are cultured at 2×10⁶ cells/ml, in RPMI-1640media supplemented with 10% decomplemented human AB serum (Biowhittaker,Walkersville, Md.), 2 mmol/liter L-glutamine, 100 U/ml penicillin G and100 μg/ml streptomycin (GIBCO BRL, Gaithersburg, Md.), in a 5% CO₂atmosphere at 37° C. To isolate monocytes, PBMC underwent plasticadherence on T175 tissue flasks (Coming-Costar, Cambridge, Mass.). Togenerate enriched populations of monocyte-derived macrophages(macrophages) and monocyte-derived dendritic cells (DCs) the followingprocedures are performed. To generate macrophages, adherent cells areextensively washed and maintained for 24 hours in medium supplementedwith 10% heat-inactivated human serum. Adherent monocytes are washed,removed from the flask by gentle scraping, seeded onto 24-well plates ata density of 1×10⁶ cells/well, and cultured for seven days. To generateimmature DCs, plastic-adhered monocytes are cultured in GM-CSF, 800 U/mland IL-4, 500 U/ml (R & DSystems, Inc., Minneapolis, Minn.) for 5 days,adding fresh GM-CSF and IL-4 on day 3. All cell culture reagents areendotoxin free.

Virus transduction and flow cytometry: Immature DCs or macrophages aretransduced at day 6. One million macrophages or DCs are incubated with50 ng p27 equivalents of scSIV (MOI of 0.05) in 100 μl volume for 2hours at 37° C. Cultures are then expanded to a volume of 2 ml in RPMIsupplemented with 5% human serum, L-glutamine, penicillin andstreptomycin and incubated at 37° C. for 4 days. The culturesupernatants of transduced macrophages or DCs are collected at varioustime points and stored at −80° C. Macrophages are stained on the plates,while DCs are harvested by gently resuspending the cells and stainingwith anti-CD40, anti-CD80, anti-CD83, anti-CD86, anti-CD11c oranti-HLA-DR or anti-CCR7 in fluorescence-activated cell sorter buffer(PBS supplemented with 3% fetal calf serum and 0.02% sodium azide).Intracellular staining for p27 is also performed to measure infectivity.Expression is monitored by flow cytometric analysis using a LSRIIbioanalyzer (Becton Dickinson) and analyzed using the FlowJo softwareprogram (Tree Star, San Carlos, Calif.).

Chemokine and cytokine assays: Cell culture supernatants are obtainedfrom macrophages and DCs infected with different viruses at various timepoints. Supernatant samples are collected, centrifuged for 5 minutes at13,000×g to clarify, and the supernatant stored at −80° C.Concentrations of IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNFα aremeasured using cytometric bead array (CBA) (BD Biosciences, San Jose,Calif.) according to the manufacturers instructions.

RT-PCR analysis of chemokine mRNA: For the measurement of MIP-1α (CCL3),MIP-1β (CCL4), and RANTES (CCL5) mRNA levels in the infected macrophagesand DCs, quantitative RT-PCR is performed. Briefly, total RNA isprepared using the RNeasy kit (Qiagen Inc., Valencia, Calif.), andreverse transcribed in a 20 μl reaction containing 0.1 μg of total RNA,0.1 μg of oligo(dT), 200 U of reverse transcriptase (Finnzymes, Finland)and 0.2 μM each of dATP, dCTP, dGTP and dTTP. After 1 hr incubation at40° C., cDNA products are generated. Real-time PCR is performed usingthe Power SYBR Green Supermix (Applied Biosystems) and the followingprimers: MIP-1α (CCL3)-specific primers, 5′-GTC TGT GCT GAT CCC AGTGA-3′ (forward) (SEQ ID NO: 5) and 5′-TTG TCA CCA GAC GCG GTG TG-3′(reverse) (SEQ ID NO: 6); MIP-1β (CCL4)-specific primers, 5′-GTC TGT GCTGAT CCC AGT GA-3′ (forward) (SEQ ID NO: 7) and 5′-GGA CAC TTA TCC TTTGGC TA-3′ (reverse) (SEQ ID NO: 8); RANTES (CCL5)-specific primers,5′-CCG CGG CAG CCC TCG CTG TCA TCC-3′ (forward) (SEQ ID NO: 9) and5′-CAT CTC CAA AGA GTT GAT GTA CTC C-3′ (reverse) (SEQ ID NO: 10). Fornormalization, GAPDH and β-actin real-time PCR is carried out on thesame samples. Normalized mRNA levels for each transcript are calculatedas (1/2ΔCt×1,000), where ΔCt value=Ct (test mRNA)-Ct (GAPDH mRNA). Tocontrol for contamination with genomic DNA, parallel amplifications areperformed in the absence of reverse transcriptase. These are uniformlynegative.

Enzyme Linked Immuno-Spot (ELISPOT) assay: IFN-γ ELISPOT assays areperformed as follows. Briefly, isolated PBMCs are plated at aconcentration of 100,000 cells per well in 96-well multiscreen plates(Millipore, Bedford, Mass.) that had been precoated with 0.5 μg/ml ofanti-IFN-γ monoclonal antibody (BD Biosciences, San Jose, Calif.). AnSIVmac239 Gag peptide pool (15-mers overlapping by 11 amino acids (NIHAIDS Reagent Program)) is added at a final concentration of 5 μg/ml.Four wells containing PBMCs and complete medium alone are used asnegative controls along with four positive controls with PhorbolMyristate Acetate (PMA, 5 ng/ml) and Ionomycin (500 ng/ml). Plates areincubated overnight at 37° C., 5% CO₂ and developed. The numbers ofspots per well are counted using an automated ELISPOT plate reader (CTLtechnologies), and the number of specific spot-forming cells (SFC), iscalculated by subtracting the negative-control wells (mean plus 3standard deviations). A value of 55 SFC/10⁶ PBMC or greater (aftersubtraction of background) is considered positive.

Statistics. Data are analyzed using PRISM 4.0 (GraphPad Software, LaJolla, Calif.) and expressed as the mean±SEM. Statistical comparisonsare analyzed by Student's t test. A p-value of 0.05 is chosen forstatistical significance.

EXAMPLE 15 Transduction of Human DCs and Macrophages with SIV EncodingLMP1 and LMP1-CD40 Results in Enhanced Activation and Maturation

Preparation of LMP1 and LMP1-CD40: Both LMP1 and LMP1-CD40 chimera genesare constructed from PCR fragments, using Raji B cell line cDNA andhuman CD40 cDNA as PCR templates. The resulting proteins are depicted inFIG. 1. The LMP1 N-terminal residues form a domain with sixtransmembrane regions that self-associates in the plane of the membrane,clustering the cytoplasmic tails of the protein. The cytoplasmic tail,either from LMP1 or CD40, contains signaling domains that recruitadapter molecules such as TRAFs to initiate downstream signaling events.Receptor-ligand interaction is not required to induce clustering, and asa result both LMP1 and LMP1-CD40 are constitutively active.

Generation of pseudotyped single-cycle SIV expressing LMP1 or LMP1-CD40:The single-cycle SIV viral construct scSIVmac239FS-ΔPRΔINEGFP is used asa template to generate single-cycle SIV virus expressing either LMP1 orLMP1-CD40 (FIG. 19). After confirming recombinant clones by sequencing,Western blot analysis is performed for Gag, LMP1, and CD40 followingtransfection of 293T cell lysates with SIV viral constructs. Gag (p27)is present in all 293T lysates, whereas LMP1 and CD40 proteins arepresent only for LMP1 and LMP1-CD40 adjuvanted viruses, respectively(FIG. 20A). Theoretical molecular weights of LMP1 (42 kDa) and LMP1-CD40(28 kDa), are consistent with Western blot values (40 kDa and 30 kDarespectively).

Transduction of human DCS and macrophages with SIV encoding LMP1 andLMP1-CD40 results in enhanced activation and maturation. Virusesexpressing LMP1, LMP1-CD40, or control GFP are tested for their abilityto activate human DCS and macrophages. The optimal infectious dose isdetermined as MOI of 0.05 and optimal time for analysis as 4 days postinfection (FIG. 27). Under these conditions, scSIV expressing LMP1 orLMP1-CD40 induces morphological changes in DCs and macrophages,including clumping and elongation of cells within the culture. Similarmorphological responses are also observed after treatment with LPS,suggesting that LMP1 and LMP1-CD40 are inducing activation of cellswithin the infected cultures. The expression levels of variousmaturation and activation surface markers on virus-transducedmacrophages and DCs are tested by flow cytometry. Cells are againevaluated 4 days after infection with scSIV viruses. Transduction withscSIV-LMP1 results in dendritic cell activation and maturation asmeasured by significantly increased levels of CD40, CD80 and CD83expression, while scSIV-LMP1-CD40 results in significant increasedlevels of CD40, CD80 and HLA-DR expression when compared toscSIV-GFP-transduced cells. (FIG. 21A). The present invention providesthat the activation signal provided by LMP1 and LMP1-CD40 is strongenough to initiate both activation and maturation of DCs. Similarly,there is a significant increase in the expression of maturation markersCD40 and CD80 on scSIV-LMP1 transduced macrophages, whereasscSIV-LMP1-CD40 results in an increase in the expression levels of CD40,CD80 and CD83 (FIG. 21B).

scSIV expressing LMP1 or LMP1-CD40 results in increased secretion ofinflammatory cytokines and β-chemokines from human and macaque DCs andmacrophages. The secretion of various human inflammatory cytokines byvirus-infected DCs or macrophages is evaluated. Inflammatory cytokineassays are performed by cytometric bead array (CBA). DCs are infectedwith different single-cycle SIV viruses at MOI of 0.05 and supernatantsare collected at various time intervals. scSIV-LMP1 infection results ina significant increase in IL-1β, IL-6, IL-8, IL-10, IL-12p70 and TNFα,while scSIV-LMP1-CD40 infection results in increase in IL-1β, IL-6,IL-8, IL-10 and TNFα at various time points (FIG. 24A). Moreover, nomeasurable amount of IL-12p70 is detected in scSIV-GFP orscSIV-LMP1-CD40 infected DCs.

Table 1 -Statistical overview of cytokines secretion from arepresentative experiment of infected human DCs and macrophages (upperpanel) and macaques DCs and macrophages (lower panel) with LMP1 andLMP1-CD40 adjuvanted virus. Human inflammatory cytokine quantitation isperformed from the culture supernatants by cytometric bead array (CBA).Data are analyzed with the unpaired t test: *, p<0.05; **, p<0.01; ***,p<0.001 compared with the scSIV-GFP infected group.

TABLE 1 Statistical overview of cytokines secretion MacrophagesDendritic Cells Cytokines SIV-LMP1- SIV-LMP1- (pg/ml) SIV-EGFP SIV-LMP1CD40 SIV-EGFP SIV-LMP1 CD40 Human IL-1β 1.53 ± 0.66 1.92 ± 0.84 ND 1.10± 0.73  8.54 ± 0.63 2.01 ± 0.62 cells  (p < 0.001) IL-6 3.53 ± 1.8427.86 ± 4.2  17.21 ± 15.2  3.66 ± 2.02 265.57 ± 66.45 13.79 ± 0.55  (p <0.01) (p < 0.01) (p < 0.01) IL-8 1882.3 ± 335   4292.5 ± 646   5000 ±0.66  537.5 ± 213.3 2941.3 ± 338.1 2330.5 ± 39.4  (p < 0.05)  (p <0.001) (p < 0.01)  (p < 0.001) IL-10 3.66 ± 0.11 2.96 ± 0.68 3.06 ± 0.314.53 ± 0.31 27.03 ± 6.64 9.57 ± 0.93 (p < 0.05) (p < 0.01) IL-12p70 ND1.63 ± 0.17 ND ND  55.8 ± 17.57 ND TNF 0.46 ± 0.42 3.13 ± 0.11 0.93 ±0.55 0.43 ± 0.57 208.39 ± 42.53 13.46 ± 1.15  (p < 0.01) (p < 0.01)  (p< 0.001) Macaque IL-1β 0.13 ± 0.04 34.30 ± 1.13  1.43 ± 0.11 0.20 ± 0.06 2.90 ± 0.80 0.76 ± 0.82 cells (p < 0.01) (p < 0.05) (p < 0.05) IL-61.90 ± 0.06 15.00 ± 2.60  7.16 ± 1.02 2.36 ± 0.37 173.63 ± 21.91 89.50 ±24.60 (p < 0.01) (p < 0.01)  (p < 0.001) (p < 0.01) IL-8 64.23 ± 2.57 1445.2 ± 158.7  1107.1 ± 99.5  2626.6 ± 537.2   5001 ± 0.66 5001 ± 0.66  (p < 0.001)  (p < 0.001) (p < 0.01) (p < 0.01) IL-10 0.40 ± 0.53 ND0.40 ± 0.53 0.50 ± 0.66 ND 0.36 ± 0.48 IL-12p70 0.83 ± 0.48 0.80 ± 0.460.80 ± 0.46 ND ND ND TNF 0.76 ± 0.44 13.50 ± 2.73  3.93 ± 0.84 1.60 ±0.20 294.12 ± 26.15 99.53 ± 33.31 (p < 0.01) (p < 0.05)  (p < 0.001) (p< 0.05)

Table 1 summarizes the concentration and p-values for cytokinessecretion from infected human and macaque DCs and macrophages (data areanalyzed with the unpaired t test: *, p<0.05; **, p<0.01; ***, p<0.001compared with the SIV-GFP infected group. ND: not detected). Values forscSIV-LMP1 or scSIV-LMP1-CD40 are compared to scSIV-GFP. Significantlyhigher secretion of inflammatory cytokines is observed from macaque DCsand macrophages upon infection with LMP1 and LMP1-CD40 adjuvanted scSIVviruses compared to control virus. In all assays LPS is used as apositive control and induced high levels of IL-8, 1L-6, and TNFα fromboth dendritic cells and macrophages. These results confirm that LMP1and LMP1-CD40 are able to activate DCs and macrophages in vitro both inhumans and non-human primates. The present invention provides thatincorporating LMP1 and LMP1-CD40 into SIV enhances its ability toactivate DCs and macrophages. β-chemokine RNA expression is alsoevaluated by real time RT-PCR of macrophages 4 days following infection.Total cellular RNA is isolated, reverse transcribed to cDNA and MIP-1α,MIP-1β, and RANTES mRNA expression is analyzed by real time PCR assay.When macrophages are infected with recombinant scSIV viruses, LMP1results in a significant increase in MIP-1β and RANTES mRNA expression,whereas LMP1-CD40 results in significant increase in MIP-1α, MIP-1β andRANTES mRNA expression (FIG. 22B). The present invention provides thatexpressions of both pro-inflammatory cytokines and β-chemokines areenhanced by single cycle SIV expressing LMP1 or LMP1-CD40.

SIV-LMP1 infected DCs can enhance antigen-specific immune responses fromautologous T cells. IL-12p70 is an important regulator of IFN-γsecretion by T cells. The present invention provides that inducedIL-12p70 production by scSIV-LMP1 transduced DCs can also increase IFN-γsecretion by autologous T cells following DC stimulation, for example,in a 12-day DC:T cell co-culture assay. DCs are transduced withscSIV-LMP1, scSIV-LMP1-CD40 or scSIV for 4 days, washed, and thencultured with autologous T cells for 12 days in the presence ofnevirapine and 5 u/ml of IL-2 (FIG. 23A). T cells are then restimulatedwith an SIV Gag 15-mer overlapping peptide pool (NIH AIDS reagentprogram). IFN-'y secreting cells are identified by ELISPOT analysis. ThescSIV-LMP1 and scSIV-LMP1-CD40 infected DCs induce an increased IFN-γ Tcell response as compared to the scSIV control (FIG. 23B).

In the present example, methods to develop safe and efficacious SIVvaccines are provided by incorporating adjuvant genes LMP1 and LMP1-CD40into the genome of single cycle SIV. These and similar vaccinationstrategies are based on the activation of DCs and macrophages via CD40signaling, resulting in an inflammatory response that is able to enhanceantigen-specific T cell responses in the vaccinee. This CD40 signalingmay be especially critical in eliciting CTL responses in conditions suchas AIDS where the number or activity of CD430 T cells is limiting. Theincorporation of LMP1 and LMP1-CD40 into scSIV viral particles resultsin enhanced immunogenicity compared to parent scSIV as evidenced by theinduction of Th1 cytokines and both DC and macrophage maturation. ThescSIV viral genome is an efficient vector for the expression of LMP1,LMP1-CD40 and the synthesis of these virus-encoded proteins is confirmedby Western blot. As an indication of the potency of the LMP1 adjuvants,scSIV viruses expressing LMP1 and LMP1-CD40 induce morphological changesin DCs and macrophages, including clumping and elongation suggestive ofactivation of these cells.

The immunogenicity of scSIV incorporating LMP1 and LMP1-CD40 isevaluated in vitro by measuring the expression levels of cell surfacemarkers transduced DCs and macrophages. The expression levels ofmaturation markers CD40, CD80, CD83, CCR7 and HLA-DR are higher in LMP1and LMP1-CD40 adjuvanted scSIV transduced DCs as compared to the GFPcontrol group. The expression of CD40, CD80, CCR7, and HLA-DR aresimilar to positive control cells matured with cytokines, however theexpression level of CD83 on LMP1 and LMP1-CD40 virus transduced DC isnot as high as that on cytokine cocktail-maturated DCs. This could beexplained by the fact that the MOI used in the infection experiments isvery low (0.05). This low MOI can lead to a low level of transfection,normally 10-20% of cells exposed to SIV constructs. These resultssuggest that the activation signal provided by LMP1 and LMP1-CD40 ispotent enough to initiate activation. There is also a modest increase inthe expression of maturation markers such as CD40 and CD83 in LMP1adjuvanted scSIV transduced macrophages, whereas LMP1-CD40 adjuvantedscSIV results in a marked increase in the expression levels of CD40,CD80 and CD83 on infected macrophages. These differences suggest apositive feedback whereby CD40 signaling from LMP1-CD40 enhances theexpression of CD40 protein on the cell surface.

Overall, Th1 cytokine secretion is dramatically enhanced by SIV encodingLMP1, with increased cytokine secretion observed within 12-24 hourspost-infection. This rapid cytokine induction includes a modest level ofIL-12p70, suggesting that SIV-LMP1 infected DCs can potentially enhanceSIV-specific T cell response in vivo. This is balanced with a modestsecretion of IL-10 following scSIV-LMP1 infection of DC. Much greatersecretion levels are observed with cytokines IL-1β, IL-6, TNFα andespecially IL-8. Transduction with SIV-LMP1 resulted in a 50-foldinduction of IL-12p70 secretion compared to transduction with SIV-GFP(from ˜1 pg/ml to 50 pg/ml at 84 hours). Given the critical role ofIL-12 in the stimulation of IFN-γ production, proliferation of T cells,and generation of cytotoxic T lymphocytes, using LMP1 as an adjuvant canresult in increased DC activation and an enhanced Th1 immune response.This IL-12 induction is consistent with LMP1 inducing a constitutiveCD40-like signal, a key role in Epstein Barr virus pathogenesis. Bindingof CD40L to its receptor on immature DCs triggers DCs activation andmaturation and increases DCs survival. One of the cytokines upregulatedin DCs activated by CD40L binding is IL-12, a cytokine responsible forpolarizing CD4+T cells to a Th1 phenotype. Researches with DNA vaccineshave showed that increasing the activation level of DC throughCD40-CD40L interactions significantly enhances the intensity of cellmediated immunity and humoral immune responses. Since IL-12 stimulatesIFN-γ production, proliferation of T cells, and generation of cytotoxicT lymphocytes, the present invention provides that LMP1 and LMP1-CD40result in increased DCs activation and a strong Th1 immune response.

The chemokines MIP-1α, MIP-1β, and RANTES play a critical role in innateimmune control of HIV by DCs and macrophages. Surprisingly, LMP1 andLMP1-CD40 are able to enhance these chemokines in the context ofrecombinant SIV virus infection. However, LMP1 is unique in its abilityto induce IL-12p70, suggesting LMP1 can be a better inducer of T cellresponses. Again, this chemokine secretion highlights the ability ofLMP1 and LMP1-CD40 to increase the immune response during SIV infectionof DCs and macrophages and suggests that these recombinant viruses mayblock viral replication while simultaneously enhances anti-HIV or SIVimmune responses.

In addition to DC maturation and cytokine secretion, the immunogenicityof LMP1 and LMP1-CD40 are further confirmed by the coculture of virusinfected DCs with autologous T cells for 12 days. The data suggests thatthe LMP1 adjuvant gene cassette is able to convert a weakly immunogenicvirus into a strongly immunogenic one that can augment T cell responsesagainst viral antigens. By comparison, scSIV-LMP1-CD40 is less active inthis assay, consistent with the overall weaker effect of scSIV-LMP1-CD40in DCs compared to scSIV-LMP1 virus.

Thus, the present invention provides that LMP1 or LMP1-CD40 can act as apotent molecular adjuvant, providing a new class of adjuvant for use inrecombinant vaccine strategies. In addition, LMP1 and LMP1chimeras/fusion proteins can be used as viral vector vaccine adjuvantsor adjuvants for DNA or RNA based vaccines. Use of LMP1 for these orother subunit vaccine strategies is provided. These vaccines canpotentially targetDCs, macrophages, and B cells, where B cell responsesare also augmented by T, LMP1 expression, including the induction of Tcell independent class switching.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the invention asdefined by the appended claims. Moreover, the scope of the presentapplication is not intended to be limited to the particular embodimentsof the process, machine, manufacture, composition of matter, means,methods and steps described in the specification. As one will readilyappreciate from the disclosure, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized. Accordingly, the appended claims areintended to include within their scope such processes, machines,manufacture, compositions of matter, means, methods, or steps.

Although the invention has been described with reference to the aboveexample, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

What is claimed is:
 1. A method for stimulating an immune response in asubject comprising: administering to a cell an effective amount of apolynucleotide comprising a first expression cassette for expressing aprotein comprising a multimerizing domain operatively joined to acytoplasmic signaling domain of a receptor such that the proteinassembles into a complex of three or more protein moieties, and/or asecond expression cassette for expressing an antigen, therebystimulating an immune response.
 2. The method of claim 1, wherein atleast two nucleic acid sequences are administered, where a first nucleicacid sequence comprises the first expression cassette for expressing theprotein comprising a multimerizing domain operatively joined to acytoplasmic signaling domain of a receptor such that the proteinassembles into a complex of three or more protein moieties, and a secondnucleic acid sequence comprises the second expression cassette forexpressing an antigen, and the two nucleic acid sequences are containedwithin the same or two different polynucleotide molecules.
 3. The methodof claim 1, wherein the nucleic acid comprises a DNA vaccine.
 4. Themethod of claim 1, wherein the nucleic acid comprises an in vitrosynthesized and optionally modified RNA molecule.
 5. The method of claim1, wherein the protein comprises a multimerizing domain comprising anN-terminus fragment of latent membrane protein 1 (LMP1); and/or acytoplasmic signaling domain comprising a cytoplasmic fragment of amember of Tumor necrosis factor receptor superfamily (TNFRSF).
 6. Themethod of claim 1, wherein the protein comprises a Latency MembraneProtein 1 (LMP1) or LMP1-CD40 fusion protein.
 7. The method of claim 1,wherein the antigen comprises a tumor antigen, viral antigen, ormicrobial antigen.